Methods and compositions for antibody-evading virus vectors

ABSTRACT

The present invention provides AAV capsid proteins comprising a modification in the amino acid sequence and virus vectors comprising the modified AAV capsid protein. The invention also provides methods of administering the virus vectors and virus capsids of the invention to a cell or to a subject in vivo.

STATEMENT OF PRIORITY

This application is a 35 U.S.C. § 371 national phase application ofInternational Application Serial No. PCT/US2016/054143, filed Sep. 28,2016, which claims the benefit, under 35 U.S.C. § 119(e), of U.S.Provisional Application No. 62/234,016, filed Sep. 28, 2015, the entirecontents of each of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government funding under Grant Nos.HL112761, HL089221 and GM082946 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5470-752_ST25.txt, 344,842 bytes in size, generated onMay 24, 2019 and filed via EFS-Web, is provided in lieu of a paper copy.This Sequence Listing is incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to modified capsid proteins fromadeno-associated virus (AAV) and virus capsids and virus vectorscomprising the same. In particular, the invention relates to modifiedAAV capsid proteins and capsids comprising the same that can beincorporated into virus vectors to confer a phenotype of evasion ofneutralizing antibodies without decreased transduction efficiency.

BACKGROUND OF THE INVENTION

Host-derived pre-existing antibodies generated upon natural encounter ofAAV or recombinant AAV vectors prevent first time as well as repeatadministration of AAV vectors as vaccines and/or for gene therapy.Serological studies reveal a high prevalence of antibodies in the humanpopulation worldwide with about 67% of people having antibodies againstAAV1, 72% against AAV2, and about 40% against AAV5 through AAV9.

Furthermore, in gene therapy, certain clinical scenarios involving genesilencing or tissue degeneration may require multiple AAV vectoradministrations to sustain long term expression of the transgene. Tocircumvent these issues, recombinant AAV vectors which evade antibodyrecognition (AAVe) are required. This invention will help a) expand theeligible cohort of patients suitable for AAV-based gene therapy and b)allow multiple, repeat administrations of AAV-based gene therapyvectors.

The present invention overcomes previous shortcomings in the art byproviding methods and compositions comprising an adeno-associated virus(AAV) capsid protein, comprising one or more amino acid substitutions,wherein the substitutions introduce into an AAV vector comprising thesemodified capsid proteins the ability to evade host antibodies.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an adeno-associated virus(AAV) capsid protein, comprising one or more amino acids substitutions,wherein the substitutions modify one or more previously existingantigenic sites on the AAV capsid protein.

In some embodiments, the amino acid substitutions are in antigenicfootprints identified by peptide epitope mapping or cryo-electronmicroscopy studies of AAV-Antibody complexes containing capsids based onAAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, AAV12,AAVrh8, AAVrh10, AAVrh32.33, Avian AAV or Bovine AAV.

In some embodiments, the modified antigenic site can prevent antibodiesfrom binding or recognizing or neutralizing AAV capsids, wherein theantibody is an IgG (including IgG1, IgG2a, IgG2b, IgG3), IgM, IgE orIgA.

In some embodiments, the modified antigenic site can prevent binding orrecognition or neutralization of AAV capsids by antibodies fromdifferent animal species, wherein the animal is human, canine, feline orequine.

In some embodiments, the modified antigenic site is a common antigenicmotif, wherein a specific antibody or a cross-reactive antibody canbind, recognize or neutralize the AAV capsid.

In some embodiments, the substitutions introduce a modified antigenicsite from a first AAV serotype into the capsid protein of a second AAVserotype that is different from said first AAV serotype.

The present invention also provides an AAV capsid comprising the AAVcapsid protein of this invention. Further provided herein is a viralvector comprising the AAV capsid of this invention as well as acomposition comprising the AAV capsid protein, AAV capsid and/or viralvector of this invention in a pharmaceutically acceptable carrier.

The present invention additionally provides a method of introducing anucleic acid into a cell in the presence of antibodies against the AAVcapsid, comprising contacting the cell with the viral vector of thisinvention. The cell can be in a subject and in some embodiments, thesubject can be a human subject.

These and other aspects of the invention are addressed in more detail inthe description of the invention set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Methods for generating AAVe strains through structuraldetermination of common antigenic motifs (CAMs) listed in Table 5 andthe generation of antibody evading AAV capsids (AAVe) by rational orcombinatorial engineering of antigenic motifs followed by amplificationand selection.

FIG. 2. Generation of AAVe libraries by random mutagenesis of amino acidresidues within common antigenic motifs (CAMs) listed in Table 5.Theoretical diversities of different libraries generated by randomizingthe different amino acid residues within each common antigenic motif.Successful generation of AAV1e libraries was confirmed via DNAsequencing of the AAV1e plasmids (SEQ ID NOS:439-442). Black solid barrepresents the position of the randomized sequences of different AAV1elibraries. Theoretical diversities were calculated by the followingequation: Theoretical diversities=20{circumflex over ( )}n, where n isthe number of randomized amino acids within the indicated CAM. FIG. 2discloses SEQ ID NO:494.

FIG. 3. In vitro antibody neutralization assay of AAV1e-series.Transduction efficiency was measured by luciferase activity. AAV1 (farleft) is neutralized by both 4E4 (top) and 5H7 (bottom) and the 50%inhibition concentration of the two antibodies are <1:64000 and 1:16000respectively. 4E4 and 5H7 are antibodies that neutralize parental AAV1.Clone AAV1e6 (middle left) is completely resistant to 4E4 neutralization(no reduction in transduction level at the highest antibodyconcentration) and partially resistant to 5H7 (50% inhibitionconcentration reduced to 1:4000). Clone AAV1e8 (middle right) showedcomplete resistance to both 4E4 and 5H7 neutralization where the highestantibody concentration showed no effect on the transduction level.AAV1e9 (far right) showed resistance to 5H7; however, it is as sensitiveto 4E4 as AAV1.

FIG. 4. In vivo antibody neutralization assay of AAV1e-series at 4 weekspost-injection into skeletal muscle of mice. Representative images ofeach virus and treatment group are shown. All viruses showed a similarlevel of transduction efficiency without antibody addition. AAV1e6 andAAV1e8 show resistance to 4E4 and AAV1e9 shows resistance to 5H7. AAV1e8also shows partial resistance to 5H7. 4E4 and 5H7 are antibodies thatcompletely neutralize parental AAV1. Luciferase activities werequantified and summarized in the bar graph (AAV1 is far left; AAV1e6 ismiddle left; AAV1e8 is middle right; AAV1e9 is far right). These resultsconfirm that the AAV1e series can escape subsets of neutralizingantibodies. Other AAV strains can be subjected to this engineering andselection protocol and similar AAVe vector series can be generated fromany capsid template using this approach.

FIG. 5. In vitro antibody neutralization assay of AAV1e clones derivedby rational combination of amino acid residues obtained from AAV1e6,AAv1e8 and AAV1e9. Transduction efficiency was measured by luciferaseactivity. AAV1 (far left) is completely neutralized by both 4E4 (top)and 5H7 (bottom) as well as the human serum sample containing polyclonalantibodies. The 50% inhibition dilution of human serum sample >1:800fold dilution. 4E4 and 5H7 are antibodies that neutralize parental AAV1.Clone AAV1e18 (middle left) is partially resistant to 4E4, 5H7 as wellas human serum. Clones AAV1e19 and AAV1e20 (middle and far right) showedcomplete resistance to both 4E4 and 5H7 neutralization as well as thehuman serum sample.

FIG. 6. Native dot blot assay comparing the parental AAV1 and AAV1eclones 27, 28 and 29 derived by rational, site-specific mutagenesis ofresidues S472R, V473D and N500E within CAM regions listed in Table 5.Assay determines the ability of AAV1e clones to escape antibodydetection. ADK1a is a monoclonal antibody that detects parental AAV1capsids.

FIG. 7. ELISA assay comparing the parental AAV1 and AAV1e clones 27, 28and 29 derived by rational, site-specific mutagenesis of residues S472R,V473D and N500E within CAM regions listed in Table 5. Assay determinesthe ability of AAV1e clones to escape antibody detection. ADK1a is amonoclonal antibody that detects parental AAV1 capsids.

FIG. 8. Transduction assay showing ability of AAV1e27 clone to evadeneutralization by ADK1a, which is an anti-capsid antibody againstparental AAV1.

FIG. 9. Native dot blot assay comparing the parental AAV1 and clonesAAV1e30-36 derived by rational, multiple site-specific mutagenesiswithin the CAM regions outlined in Table 5. Assay determines the abilityof AAV1e clones to escape antibody detection. 4E4 and 5H7 are anti-AAV1capsid antibodies.

FIG. 10. Transduction assay comparing the parental AAV1 and clonesAAV1e30-36 derived by rational, multiple site-specific mutagenesiswithin the CAM regions outlined in Table 5. Assay determines the abilityof AAV1e clones to escape antibody detection. 4E4 and 5H7 are monoclonalantibodies against the parental AAV1 capsid and the human serum samplecontains polyclonal antibodies against AAV1. Clones AAV1e30-36completely escape 4E4, while parental AAV1 is neutralized. ClonesAAV1e34 and AAV1e35 show substantial ability to escape 5H7, whileAAV1e36 displays a partial ability for evading 5H7. Clone AAV1e36escapes polyclonal antibodies in a human patient serum sample (50%neutralization for parental AAV1 is 1:320 dilution, while AAV1e36 isshifted to between 1:40 and 1:80 dilution range.

FIG. 11. Native dot blot assay comparing the parental AAV9 and clonesAAV9e1 and AAV9e2 derived by rational, site-specific mutagenesis ofresidues listed within the CAM regions outlined in Table 5. Assayestablishes the ability to engineer another serotype AAV9 to evadeantibodies and the ability of AAV9e clones to escape antibody detection.ADK9, HL2368, HL2370 and HL2372 are monoclonal antibodies that detectparental AAV9 capsids.

FIGS. 12A-12D. Roadmap for structure-based evolution of antigenicallyadvanced AAV variants. (A) Three-dimensional model of cryo-reconstructedAAV1 capsid complexed to multiple monoclonal antibodies. The modeldepicts AAV1 complexed with the Fab regions of 4 different monoclonalantibodies viewed along the 2-fold axis, ADK1a, ADK1b, 4E4, 5H7. (B)Contact residues and common antigenic motifs (CAMs) for four anti-AAV1antibodies on the capsid generated by RIVEM are shown. Color codes ofeach antibody are same as above, in addition, overlapping residuesbetween antibodies were colored individually, ADK1a and 4E4, 4E4 and5H7. (C) Individual antigenic footprints on the AAV1 capsid selected forengineering and AAV library generation. Three different AAV librarieswere subjected to five rounds of evolution on vascular endothelial cellsco-infected with adenovirus to yield single region AAV-CAM variants. (D)Newly evolved antigenic footprints from each library were then combinedand re-engineered through an iterative process, pooled and subjected toa second round of directed evolution for 3 cycles. This approach yieldsantigenically advanced AAV-CAM variants with new footprints that havenot yet emerged in nature.

FIGS. 13A-13H. Analysis of library diversity, directed evolution andenrichment of novel antigenic footprints. Parental and evolved librarieswere subjected to high-throughput sequencing using the Illumina MiSeqplatform. Following analysis with a custom Perl script, enriched aminoacid sequences were plotted in R for both the parental and evolvedlibraries of (A) region 4, (B) region 5, (C) region 8 and (D) combinedregions 5+8. Each bubble represents a distinct capsid amino acidsequence with the area proportional to the number of reads for thatvariant in the respective library. (E-H) Amino acid sequencerepresentation was calculated for the top ten variants with the highestrepresentation in each library after subjecting to evolution.Percentages represent the number of reads for the variant in the evolvedlibrary normalized to the total number of reads containing the antigenicregion of interest. “Other” sequences represent all other evolvedlibrary amino acid sequences not contained in the top ten hits. FIG. 13Adiscloses SEQ ID NOs:22, 23, 25, 483 and 527. FIG. 13B discloses SEQ IDNOs:485, 501 and 502. FIG. 13C discloses SEQ ID NOs:309, 486, 510. FIG.13D discloses SEQ ID NOs:309 and 487. FIG. 13E discloses SEQ IDNOs:22-25, 483 and 495-500. FIG. 13F discloses SEQ ID NOs:485 and501-509. FIG. 13G discloses SEQ ID NOs: 32, 37, 38 and 510-515. FIG. 13Hdiscloses SEQ ID NOs:309-487 and 516-523.

FIGS. 14A-14I. Neutralization profile of AAV1 and single region CAMvariants against mouse monoclonal antibodies (MAbs) in vitro and invivo. (A-C) Different AAV strains, AAV1, CAM106, CAM108 and CAM109evaluated against MAbs 4E4, 5H7 and ADK1a at different dilutions ofhybridoma media. Relative luciferase transgene expression mediated bydifferent vectors mixed with MAbs was normalized to no antibodycontrols. Error bars represent standard deviation (n=4). (D) Roadmapimages of the 3-fold axis of each CAM mutant showing the location ofnewly evolved antigenic footprints—CAM106, CAM108 and CAM109. (E-H)Luciferase expression in mouse hind limb muscles injected with a dose of2×10¹⁰ vg of AAV1, CAM106, CAM108 and CAM109 vectors packaging ssCBA-Lucand mixed with different MAbs. Representative live animal images at 4wks post-injection are shown in the following subgroups (E) no antibodycontrol, (F) 4E4 (1:500), (G) 5H7 (1:50) and (H) ADK1a (1:5). (I)Quantitation of luciferase activity mediated by different CAM variantsrelative to parental AAV1. Luciferase activity is expressed asphotons/sec/cm2/sr as calculated by Living Image 3.2 software. Errorbars represent S.D. (n=3).

FIGS. 15A-15E. Neutralization profiles of AAV1 and CAM variants inpre-immunized mouse antisera. (A) Roadmap images of each antigenicallyadvanced CAM variant showing newly evolved footprints at the 3-foldsymmetry axis—CAM117 (regions 4+5), CAM125 (regions 5+8, cyan) andCAM130 (regions 4+5+8). (B-D) Anti-AAV1 mouse serum from threeindividual animals and (E) control mouse serum were serially diluted in2-fold increments from 1:50-1:3200 and co-incubated with AAV vectors invitro. The dotted line represents NAb-mediated inhibition of AAVtransduction by 50%. Solid lines represent relative transductionefficiencies of AAV1, CAM117, CAM125 and CAM130 at different dilutionsof antisera. Error bars represented S.D. (n=3).

FIGS. 16A-16I. Neutralization profiles of AAV1 and CAM130 in non-humanprimate antisera. Serum samples collected from three individual rhesusmacaques collected at pre-(naïve) and post-immunization (at 4 wks and 9wks) were serially diluted at 2-fold increments from 1:5-1:320 andco-incubated with AAV vectors in vitro. The dotted line representsNAb-mediated inhibition of AAV transduction by 50%. Solid linesrepresent relative transduction efficiencies of AAV1 and CAM130 atdifferent dilutions of antisera. Error bars represented S.D. (n=3).

FIGS. 17A and 17B. Neutralization profile of AAV1 and CAM130 againstindividual primate and human serum samples. AAV1 and CAM130 packagingCBA-Luc (MOI 10,000) were tested against (A) primate and (B) human seraat a 1:5 dilution to reflect clinically relevant exclusion criteria. Thedotted line represents NAb-mediated inhibition of AAV transduction by50%. Solid bars represent relative transduction efficiencies of AAV1 andCAM130. Error bars represented S.D. (n=3).

FIG. 18A-18D. In vive characterization of the CAM130 variant. Luciferasetransgene expression profiles of AAV1 and CAM130 in (A) heart and (C)liver at 2 wks post-intravenous administration of 1×10¹¹ vg/mouse (n=5).Dotted lines show background levels of luciferase activity in mockinjection controls. Biodistribution of AAV1 and CAM130 vector genomes in(B) heart and (D) liver. Vector genome copy numbers per cell werecalculated and values from mock injection controls were subtracted toobtain final values. Each dot represented a duplicated experiment from asingle animal (n=5) and the dash represents the mean value.

FIGS. 19A-19C. Physical and biological properties of CAM variantscompared to AAV1. (A) Titers of purified CAM variants produced using thetriple plasmid transfection protocol in HEK293 cells (four 150 mmculture dishes). Transduction profile of (B) single CAM variants and (C)combined CAM variants compared to AAV1 on vascular endothelial cells(MB114).

FIG. 20. Sequencing Reads Mapped to Region of Interest. Percentage ofsequencing reads mapped to the mutagenized region of interest forunselected and selected libraries CAM5, CAM8, CAM58, and CAM4.Demultiplexed FASTQ files were processed and mapped with a custom Perlscript.

FIG. 21. Representation of lead variants in unselected and selectedlibraries. Percentage representation of amino acid sequences for leadvariants in unselected and selected libraries, calculated by dividingthe reads containing a sequence of interest by the total readscontaining the mutagenized region. FIG. 21 discloses SEQ ID NOs:534-527.

FIG. 22. Transduction of human hepatocarcinoma cells Huh7 by AAV8emutants. Transduction efficiency of AAV8e mutants AAV8e01, AAV8e04 andAAV8e05 of Huh7 cells was determined and compared to the transduction ofHuh7 cells by wild-type AAV8.

FIGS. 23A-23C. Escape of AAV8e mutants from neutralization by mousemonoclonal antibodies against AAV8. The ability of AAV8e mutants toescape neutralization was examined using mAbs HL2381 (A), HL2383 (B) andADK8 (C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to theaccompanying drawings, in which representative embodiments of theinvention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, GenBank accessionnumbers and other references mentioned herein are incorporated byreference herein in their entirety.

The designation of all amino acid positions in the AAV capsid proteinsin the description of the invention and the appended claims is withrespect to VP1 capsid subunit numbering. It will be understood by thoseskilled in the art that the modifications described herein if insertedinto the AAV cap gene may result in modifications in the VP1, VP2 and/orVP3 capsid subunits. Alternatively, the capsid subunits can be expressedindependently to achieve modification in only one or two of the capsidsubunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3).

Definitions

The following terms are used in the description herein and the appendedclaims:

The singular forms “a,” “an” and “the” are intended to include theplural forms as well, unless the context clearly indicates otherwise.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of the length of a polynucleotide orpolypeptide sequence, dose, time, temperature, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

To illustrate further, if, for example, the specification indicates thata particular amino acid can be selected from A, G, I, L and/or V, thislanguage also indicates that the amino acid can be selected from anysubset of these amino acid(s) for example A, G, I or L; A, G, I or V; Aor G; only L; etc. as if each such subcombination is expressly set forthherein. Moreover, such language also indicates that one or more of thespecified amino acids can be disclaimed. For example, in particularembodiments the amino acid is not A, G or I; is not A; is not G or V;etc. as if each such possible disclaimer is expressly set forth herein.

As used herein, the terms “reduce,” “reduces,” “reduction” and similarterms mean a decrease of at least about 10%, 15%, 20%, 25%, 35%, 50%,75%, 80%, 85%, 90%, 95%, 97% or more.

As used herein, the terms “enhance,” “enhances,” “enhancement” andsimilar terms indicate an increase of at least about 10%, 15%, 20%, 25%,50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

The term “parvovirus” as used herein encompasses the familyParvoviridae, including autonomously replicating parvoviruses anddependoviruses. The autonomous parvoviruses include members of thegenera Protoparvovirus, Erythroparvovirus, Bocaparvirus, and Densovirussubfamily. Exemplary autonomous parvoviruses include, but are notlimited to, minute virus of mouse, bovine parvovirus, canine parvovirus,chicken parvovirus, feline panleukopenia virus, feline parvovirus, gooseparvovirus, H1 parvovirus, muscovy duck parvovirus, B19 virus, and anyother autonomous parvovirus now known or later discovered. Otherautonomous parvoviruses are known to those skilled in the art. See,e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed.,Lippincott-Raven Publishers; Cotmore et al. Archives of Virology DOI10.1007/s00705-013-1914-I).

As used herein, the term “adeno-associated virus” (AAV), includes but isnot limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3Aand 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAVtype 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV typerh32.33, AAV type rh8, AAV type rh10, avian AAV, bovine AAV, canine AAV,equine AAV, ovine AAV, and any other AAV now known or later discovered.See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4thed., Lippincott-Raven Publishers). A number of AAV serotypes and cladeshave been identified (see, e.g., Gao et al., (2004) J. Virology78:6381-6388; Moris et al., (2004) Virology 33-:375-383; and Table 1).

The genomic sequences of various serotypes of AAV and the autonomousparvoviruses, as well as the sequences of the native terminal repeats(TRs), Rep proteins, and capsid subunits are known in the art. Suchsequences may be found in the literature or in public databases such asGenBank. See, e.g., GenBank Accession Numbers NC_002077, NC_001401,NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701,NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705,AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226,AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; thedisclosures of which are incorporated by reference herein for teachingparvovirus and AAV nucleic acid and amino acid sequences. See also,e.g., Srivistava et al., (1983) J. Virology 45:555; Chiorini et al.,(1998) J. Virology 71:6823; Chiorini et al., (1999) J. Virology 73:1309;Bantel-Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J.Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade etal., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad Sci.USA 99:11854; Moris et al., (2004) Virology 33-:375-383; internationalpatent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat.No. 6,156,303; the disclosures of which are incorporated by referenceherein for teaching parvovirus and AAV nucleic acid and amino acidsequences. See also Table 1. The capsid structures of autonomousparvoviruses and AAV are described in more detail in BERNARD N. FIELDSet al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-RavenPublishers). See also, description of the crystal structure of AAV2 (Xieet al., (2002) Proc. Nat. Acad Sci. 99:10405-10), AAV9 (DiMattia et al.,(2012) J. Virol. 86:6947-6958), AAV8 (Nam et al., (2007) J. Virol.81:12260-12271), AAV6 (Ng et al., (2010) J. Virol. 84:12945-12957), AAV5(Govindasamy et al., (2013) J. Virol. 87, 11187-11199), AAV4(Govindasamy et al., (2006) J. Virol. 80:11556-11570), AAV3B (Lerch etal., (2010) Virology 403: 26-36), BPV (Kailasan et al., (2015) J. Virol.89:2603-2614) and CPV (Xie et al., (1996) J. Mol. Biol. 6:497-520 andTsao et al., (1991) Science 251: 1456-64).

TABLE 1 AAV Serotypes/Isolates GenBank Accession Number Clonal IsolatesAvian AAV ATCC VR-865 AY186198, AY629583, NC_004828 Avian AAV strainDA-1 NC_006263, AY629583 Bovine AAV NC_005889, AY388617 AAV4 NC_001829AAV5 AY18065, AF085716 Rh34 AY243001 Rh33 AY243002 Rh32 AY243003 Clade AAAV1 NC_002077, AF063497 AAV6 NC_001862 Hu.48 AY530611 Hu 43 AY530606 Hu44 AY530607 Hu 46 AY530609 Clade B Hu19 AY530584 Hu20 AY530586 Hu23AY530589 Hu22 AY530588 Hu24 AY530590 Hu21 AY530587 Hu27 AY530592 Hu28AY530593 Hu29 AY530594 Hu63 AY530624 Hu64 AY530625 Hu13 AY530578 Hu56AY530618 Hu57 AY530619 Hu49 AY530612 Hu58 AY530620 Hu34 AY530598 Hu35AY530599 AAV2 NC_001401 Hu45 AY530608 Hu47 AY530610 Hu51 AY530613 Hu52AY530614 Hu T41 AY695378 Hu S17 AY695376 Hu T88 AY695375 Hu T71 AY695374Hu T70 AY695373 Hu T40 AY695372 Hu T32 AY695371 Hu T17 AY695370 Hu LG15AY695377 Clade C AAV 3 NC_001729 AAV3B NC_001863 Hu9 AY530629 Hu10AY530576 Hu11 AY530577 Hu53 AY530615 Hu55 AY530617 Hu54 AY530616 Hu7AY530628 Hu18 AY530583 Hu15 AY530580 Hu16 AY530581 Hu25 AY530591 Hu60AY530622 Ch5 AY243021 Hu3 AY530595 Hu1 AY530575 Hu4 AY530602 Hu2AY530585 Hu61 AY530623 Clade D Rh62 AY530573 Rh48 AY530561 Rh54 AY530567Rh55 AY530568 Cy2 AY243020 AAV7 AF513851 Rh35 AY243000 Rh37 AY242998Rh36 AY242999 Cy6 AY243016 Cy4 AY243018 Cy3 AY243019 Cy5 AY243017 Rh13AY243013 Clade E Rh38 AY530558 Hu66 AY530626 Hu42 AY530605 Hu67 AY530627Hu40 AY530603 Hu41 AY530604 Hu37 AY530600 Rh40 AY530559 Rh2 AY243007 Bb1AY243023 Bb2 AY243022 Rh10 AY243015 Hu17 AY530582 Hu6 AY530621 Rh25AY530557 Pi2 AY530554 Pi1 AY530553 Pi3 AY530555 Rh57 AY530569 Rh50AY530563 Rh49 AY530562 Hu39 AY530601 Rh58 AY530570 Rh61 AY530572 Rh52AY530565 Rh53 AY530566 Rh51 AY530564 Rh64 AY530574 Rh43 AY530560 AAV8AF513852 Rh8 AY242997 Rh1 AY530556 Clade F AAV9 (Hu14) AY530579 Hu31AY530596 Hu32 AY530597

The term “tropism” as used herein refers to preferential entry of thevirus into certain cells or tissues, optionally followed by expression(e.g., transcription and, optionally, translation) of a sequence(s)carried by the viral genome in the cell, e.g., for a recombinant virus,expression of a heterologous nucleic acid(s) of interest.

Those skilled in the art will appreciate that transcription of aheterologous nucleic acid sequence from the viral genome may not beinitiated in the absence of trans-acting factors, e.g., for an induciblepromoter or otherwise regulated nucleic acid sequence. In the case of arAAV genome, gene expression from the viral genome may be from a stablyintegrated provirus, from a non-integrated episome, as well as any otherform in which the virus may take within the cell.

As used here, “systemic tropism” and “systemic transduction” (andequivalent terms) indicate that the virus capsid or virus vector of theinvention exhibits tropism for or transduces, respectively, tissuesthroughout the body (e.g., brain, lung, skeletal muscle, heart, liver,kidney and/or pancreas). In embodiments of the invention, systemictransduction of muscle tissues (e.g., skeletal muscle, diaphragm andcardiac muscle) is observed. In other embodiments, systemic transductionof skeletal muscle tissues achieved. For example, in particularembodiments, essentially all skeletal muscles throughout the body aretransduced (although the efficiency of transduction may vary by muscletype). In particular embodiments, systemic transduction of limb muscles,cardiac muscle and diaphragm muscle is achieved. Optionally, the viruscapsid or virus vector is administered via a systemic route (e.g.,systemic route such as intravenously, intra-articularly orintra-lymphatically). Alternatively, in other embodiments, the capsid orvirus vector is delivered locally (e.g., to the footpad,intramuscularly, intradermally, subcutaneously, topically).

Unless indicated otherwise, “efficient transduction” or “efficienttropism,” or similar terms, can be determined by reference to a suitablecontrol (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or moreof the transduction or tropism, respectively, of the control). Inparticular embodiments, the virus vector efficiently transduces or hasefficient tropism for skeletal muscle, cardiac muscle, diaphragm muscle,pancreas (including n-islet cells), spleen, the gastrointestinal tract(e.g., epithelium and/or smooth muscle), cells of the central nervoussystem, lung, joint cells, and/or kidney. Suitable controls will dependon a variety of factors including the desired tropism profile. Forexample, AAV8 and AAV9 are highly efficient in transducing skeletalmuscle, cardiac muscle and diaphragm muscle, but have the disadvantageof also transducing liver with high efficiency. Thus, the invention canbe practiced to identify viral vectors of the invention that demonstratethe efficient transduction of skeletal, cardiac and/or diaphragm muscleof AAV8 or AAV9, but with a much lower transduction efficiency forliver. Further, because the tropism profile of interest may reflecttropism toward multiple target tissues, it will be appreciated that asuitable vector may represent some tradeoffs. To illustrate, a virusvector of the invention may be less efficient than AAV8 or AAV9 intransducing skeletal muscle, cardiac muscle and/or diaphragm muscle, butbecause of low level transduction of liver, may nonetheless be verydesirable.

Similarly, it can be determined if a virus “does not efficientlytransduce” or “does not have efficient tropism” for a target tissue, orsimilar terms, by reference to a suitable control. In particularembodiments, the virus vector does not efficiently transduce (i.e., hasdoes not have efficient tropism) for liver, kidney, gonads and/or germcells. In particular embodiments, undesirable transduction of tissue(s)(e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1%or less of the level of transduction of the desired target tissue(s)(e.g., skeletal muscle, diaphragm muscle, cardiac muscle and/or cells ofthe central nervous system).

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “polynucleotide” is a sequence of nucleotide bases, and may be RNA,DNA or DNA-RNA hybrid sequences (including both naturally occurring andnon-naturally occurring nucleotide), but in representative embodimentsare either single or double stranded DNA sequences.

As used herein, an “isolated” polynucleotide (e.g., an “isolated DNA” oran “isolated RNA”) means a polynucleotide at least partially separatedfrom at least some of the other components of the naturally occurringorganism or virus, for example, the cell or viral structural componentsor other polypeptides or nucleic acids commonly found associated withthe polynucleotide. In representative embodiments an “isolated”nucleotide is enriched by at least about 10-fold, 100-fold, 1000-fold,10,000-fold or more as compared with the starting material.

Likewise, an “isolated” polypeptide means a polypeptide that is at leastpartially separated from at least some of the other components of thenaturally occurring organism or virus, for example, the cell or viralstructural components or other polypeptides or nucleic acids commonlyfound associated with the polypeptide. In representative embodiments an“isolated” polypeptide is enriched by at least about 10-fold, 100-fold,1000-fold, 10,000-fold or more as compared with the starting material.

As used herein, by “isolate” or “purify” (or grammatical equivalents) avirus vector, it is meant that the virus vector is at least partiallyseparated from at least some of the other components in the startingmaterial. In representative embodiments an “isolated” or “purified”virus vector is enriched by at least about 10-fold, 100-fold, 1000-fold,10,000-fold or more as compared with the starting material.

A “therapeutic polypeptide” is a polypeptide that can alleviate, reduce,prevent, delay and/or stabilize symptoms that result from an absence ordefect in a protein in a cell or subject and/or is a polypeptide thatotherwise confers a benefit to a subject, e.g., anti-cancer effects orimprovement in transplant survivability.

By the terms “treat,” “treating” or “treatment of” (and grammaticalvariations thereof) it is meant that the severity of the subject'scondition is reduced, at least partially improved or stabilized and/orthat some alleviation, mitigation, decrease or stabilization in at leastone clinical symptom is achieved and/or there is a delay in theprogression of the disease or disorder.

The terms “prevent,” “preventing” and “prevention” (and grammaticalvariations thereof) refer to prevention and/or delay of the onset of adisease, disorder and/or a clinical symptom(s) in a subject and/or areduction in the severity of the onset of the disease, disorder and/orclinical symptom(s) relative to what would occur in the absence of themethods of the invention. The prevention can be complete, e.g., thetotal absence of the disease, disorder and/or clinical symptom(s). Theprevention can also be partial, such that the occurrence of the disease,disorder and/or clinical symptom(s) in the subject and/or the severityof onset is less than what would occur in the absence of the presentinvention.

A “treatment effective” amount as used herein is an amount that issufficient to provide some improvement or benefit to the subject.Alternatively stated, a “treatment effective” amount is an amount thatwill provide some alleviation, mitigation, decrease or stabilization inat least one clinical symptom in the subject. Those skilled in the artwill appreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

A “prevention effective” amount as used herein is an amount that issufficient to prevent and/or delay the onset of a disease, disorderand/or clinical symptoms in a subject and/or to reduce and/or delay theseverity of the onset of a disease, disorder and/or clinical symptoms ina subject relative to what would occur in the absence of the methods ofthe invention. Those skilled in the art will appreciate that the levelof prevention need not be complete, as long as some benefit is providedto the subject.

The terms “heterologous nucleotide sequence” and “heterologous nucleicacid” are used interchangeably herein and refer to a sequence that isnot naturally occurring in the virus. Generally, the heterologousnucleic acid comprises an open reading frame that encodes a polypeptideor nontranslated RNA of interest (e.g., for delivery to a cell orsubject).

As used herein, the terms “virus vector,” “vector” or “gene deliveryvector” refer to a virus (e.g., AAV) particle that functions as anucleic acid delivery vehicle, and which comprises the vector genome(e.g., viral DNA [vDNA]) packaged within a virion.

Alternatively, in some contexts, the term “vector” may be used to referto the vector genome/vDNA alone.

A “rAAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA)that comprises one or more heterologous nucleic acid sequences. rAAVvectors generally require only the terminal repeat(s) (TR(s)) in cis togenerate virus. All other viral sequences are dispensable and may besupplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol.158:97). Typically, the rAAV vector genome will only retain the one ormore TR sequence so as to maximize the size of the transgene that can beefficiently packaged by the vector. The structural and non-structuralprotein coding sequences may be provided in trans (e.g., from a vector,such as a plasmid, or by stably integrating the sequences into apackaging cell). In embodiments of the invention the rAAV vector genomecomprises at least one TR sequence (e.g., AAV TR sequence), optionallytwo TRs (e.g., two AAV TRs), which typically will be at the 5′ and 3′ends of the vector genome and flank the heterologous nucleic acid, butneed not be contiguous thereto. The TRs can be the same or differentfrom each other.

The term “terminal repeat” or “TR” includes any viral terminal repeat orsynthetic sequence that forms a hairpin structure and functions as aninverted terminal repeat (i.e., mediates the desired functions such asreplication, virus packaging, integration and/or provirus rescue, andthe like). The TR can be an AAV TR or a non-AAV TR. For example, anon-AAV TR sequence such as those of other parvoviruses (e.g., canineparvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or anyother suitable virus sequence (e.g., the SV40 hairpin that serves as theorigin of SV40 replication) can be used as a TR, which can further bemodified by truncation, substitution, deletion, insertion and/oraddition. Further, the TR can be partially or completely synthetic, suchas the “double-D sequence” as described in U.S. Pat. No. 5,478,745 toSamulski et al.

An “AAV terminal repeat” or “AAV TR” may be from any AAV, including butnot limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 orany other AAV now known or later discovered (see, e.g., Table 1). An AAVterminal repeat need not have the native terminal repeat sequence (e.g.,a native AAV TR sequence may be altered by insertion, deletion,truncation and/or missense mutations), as long as the terminal repeatmediates the desired functions, e.g., replication, virus packaging,integration, and/or provirus rescue, and the like.

The virus vectors of the invention can further be “targeted” virusvectors (e.g., having a directed tropism) and/or a “hybrid” parvovirus(i.e., in which the viral TRs and viral capsid are from differentparvoviruses) as described in international patent publication WO00/28004 and Chao et al., (2000) Molecular Therapy 2:619.

The virus vectors of the invention can further be duplexed parvovirusparticles as described in international patent publication WO 01/92551(the disclosure of which is incorporated herein by reference in itsentirety). Thus, in some embodiments, double stranded (duplex) genomescan be packaged into the virus capsids of the invention.

Further, the viral capsid or genomic elements can contain othermodifications, including insertions, deletions and/or substitutions.

As used herein, the term “amino acid” encompasses any naturallyoccurring amino acid, modified forms thereof, and synthetic amino acids.

Naturally occurring, levorotatory (L-) amino acids are shown in Table2).

TABLE 2 Abbreviation Amino Acid Residue Three-Letter Code One-LetterCode Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid(Aspartate) Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid(Glutamate) Glu E Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

Alternatively, the amino acid can be a modified amino acid residue(nonlimiting examples are shown in Table 3) and/or can be an amino acidthat is modified by post-translation modification (e.g., acetylation,amidation, formylation, hydroxylation, methylation, phosphorylation orsulfatation).

TABLE 3 Modified Amino Acid Residue Abbreviation Amino Acid ResidueDerivatives 2-Aminoadipic acid Aad 3-Aminoadipic acid bAad beta-Alanine,beta-Aminoproprionic acid bAla 2-Aminobutyric acid Abu 4-Aminobutyricacid, Piperidinic acid 4Abu 6-Aminocaproic acid Acp 2-Aminoheptanoicacid Ahe 2-Aminoisobutyric acid Aib 3-Aminoisobutyric acid bAib2-Aminopimelic acid Apm t-butylalanine t-BuA Citrulline CitCyclohexylalanine Cha 2,4-Diaminobutyric acid Dbu Desmosine Des2,2′-Diaminopimelic acid Dpm 2,3-Diaminoproprionic acid DprN-Ethylglycine EtGly N-Ethylasparagine EtAsn Homoarginine hArgHomocysteine hCys Homoserine hSer Hydroxylysine Hyl Allo-HydroxylysineaHyl 3-Hydroxyproline 3Hyp 4-Hydroxyproline 4Hyp Isodesmosine Ideallo-Isoleucine aIle Methionine sulfoxide MSO N-Methylglycine, sarcosineMeGly N-Methylisoleucine MeIle 6-N-Methyllysine MeLys N-MethylvalineMeVal 2-Naphthylalanine 2-Nal Norvaline Nva Norleucine Nle Ornithine Orn4-Chlorophenylalanine Phe(4-Cl) 2-Fluorophenylalanine Phe(2-F)3-Fluorophenylalanine Phe(3-F) 4-Fluorophenylalanine Phe(4-F)Phenylglycine Phg Beta-2-thienylalanine Thi

Further, the non-naturally occurring amino acid can be an “unnatural”amino acid as described by Wang et al., Annu Rev Biophys Biomol Struct.35:225-49 (2006)). These unnatural amino acids can advantageously beused to chemically link molecules of interest to the AAV capsid protein.

Modified AAV Capsid Proteins and Virus Capsids and Virus VectorsComprising the Same.

The present invention provides AAV capsid proteins (VP1, VP2 and/or VP3)comprising a modification (e.g., a substitution) in the amino acidsequence and virus capsids and virus vectors comprising the modified AAVcapsid protein. The inventors have discovered that modifications of thisinvention can confer one or more desirable properties to virus vectorscomprising the modified AAV capsid protein including without limitation,the ability to evade neutralizing antibodies. Thus, the presentinvention addresses some of the limitations associated with conventionalAAV vectors.

Accordingly, in one aspect, the present invention provides anadeno-associated virus (AAV) capsid protein, comprising one or moreamino acid substitutions, wherein the one or more substitutions modifyone or more antigenic sites on the AAV capsid protein. The modificationof the one or more antigenic sites results in inhibition of binding byan antibody to the one or more antigenic sites and/or inhibition ofneutralization of infectivity of a virus particle comprising said AAVcapsid protein. The one or more amino acid substitutions can be in oneor more antigenic footprints identified by peptide epitope mappingand/or cryo-electron microscopy studies of AAV-antibody complexescontaining AAV capsid proteins. In some embodiments, the one or moreantigenic sites are common antigenic motifs or CAMs (see, e.g., Table5). The capsid proteins of this invention are modified to produce an AAVcapsid that is present in an AAV virus particle or AAV virus vector thathas a phenotype of evading neutralizing antibodies. The AAV virusparticle or vector of this invention can also have a phenotype ofenhanced or maintained transduction efficiency in addition to thephenotype of evading neutralizing antibodies.

In some embodiments, the one or more substitutions of the one or moreantigenic sites can introduce one or more antigenic sites from a capsidprotein of a first AAV serotype into the capsid protein of a second AAVserotype that is different from said first AAV serotype.

The AAV capsid protein of this invention can be a capsid protein of anAAV serotype selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh.32.33,bovine AAV, avian AAV or any other AAV now known or later identified.

Several examples of a modified AAV capsid protein of this invention areprovided herein. In the following examples, the capsid protein cancomprise the specific substitutions described and in some embodimentscan comprise fewer or more substitutions than those described. Forexample in some embodiments, a capsid protein of this invention cancomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., substitutions.

Furthermore, in the embodiments described herein wherein an amino acidresidue is substituted by any amino acid residue other than the aminoacid residue present in the wild type or native amino acid sequence,said any other amino acid residue can be any natural or non-naturalamino acid residue known in the art (see, e.g., Tables 2 and 3). In someembodiments, the substitution can be a conservative substitution and insome embodiments, the substitution can be a nonconservativesubstitution.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acidresidues 262-268 of AAV1 (VP1 numbering; CAM1), in any combination, orthe equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33,bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) ofamino acid residues 370-379 of AAV1 (VP1 numbering; CAM 3), in anycombination, or the equivalent amino acid residues in AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10,AAVrh32.33, bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6, 7, 8 or 9) of aminoacid residues 451-459 of AAV1 (VP1 numbering; CAM 4-1), in anycombination, or the equivalent amino acid residues in AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10,AAVrh32.33, bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2) of amino acid residues 472-473 ofAAV1 (VP1 numbering; CAM 4-2) or the equivalent amino acid residues inAAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,AAVrh8, AAVrh10, AAVrh32.33, bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) of amino acidresidues 493-500 of AAV1 (VP1 numbering; CAM 5), in any combination, orthe equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33,bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acidresidues 528-534 of AAV1 (VP1 numbering; CAM 6), in any combination, orthe equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33,bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, or 6) of amino acidresidues 547-552 of AAV1 (VP1 numbering; CAM 7), in any combination, orthe equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV1, AAV12, AAVrh8, AAVrh10, AAVrh32.33,bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) ofamino acid residues 588-597 of AAV1 (VP1 numbering; CAM 8), in anycombination, or the equivalent amino acid residues in AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10,AAVrh32.33, bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2) of amino acid residues 709-710 ofAAV1 (VP1 numbering; CAM 9-1), or the equivalent amino acid residues inAAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,AAVrh8, AAVrh10, AAVrh32.33, bovine AAV or avian AAV.

In some embodiments, the capsid protein of this invention can comprise asubstitution at one or more (e.g., 2, 3, 4, 5, 6 or 7) of amino acidresidues 716-722 of AAV1 (VP1 numbering; CAM 9-2), in any combination,or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33,bovine AAV or avian AAV.

In particular embodiments of this invention, an adeno-associated virus(AAV) capsid protein is provided herein, wherein the capsid proteincomprises one or more substitution at all positions or in anycombination of fewer than all positions, resulting in the amino acidsequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:18) at the amino acidscorresponding to amino acid positions 262 to 268 (VP1 numbering) of thenative AAV1 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than A; wherein X³ is any amino acidother than S; wherein X⁴ is any amino acid other than T; wherein X⁵ isany amino acid other than G; wherein X⁶ is any amino acid other than A;and wherein X⁷ is any amino acid other than S. In embodiments whereinany of X¹ through X⁷ is not substituted, the amino acid residue at theunsubstituted position is the wild type amino acid residue.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹X¹⁰ (SEQ ID NO:19) at the aminoacids corresponding to amino acid positions 370 to 379 (VP1 numbering)of the native AAV1 capsid protein, wherein X¹ is any amino acid otherthan V; wherein X² is any amino acid other than F; wherein X³ is anyamino acid other than M; wherein X⁴ is any amino acid other than I;wherein X⁵ is any amino acid other than P; wherein X⁶ is any amino acidother than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸ isany amino acid other than G; wherein X⁹ is any amino acid other than Y;and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:20) at the aminoacids corresponding to amino acid positions 451 to 459 (VP1 numbering)of the native AAV1 capsid protein, wherein X¹ is any amino acid otherthan N; wherein X² is any amino acid other than Q; wherein X³ is anyamino acid other than S; wherein X⁴ is any amino acid other than G;wherein X⁵ is any amino acid other than S; wherein X⁶ is any amino acidother than A; wherein X⁷ is any amino acid other than Q; X¹ is any aminoacid other than N and X⁹ is any amino acid other than K. In particularembodiments, X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:21) can be: (a) QVRG (SEQ ID NO:22);(b) ERPR (SEQ ID NO:23); (c) GRGG (SEQ ID NO:24); (d) SGGR (SEQ IDNO:25); (e) SERR (SEQ ID NO:26); or (f) LRGG (SEQ ID NO:27).

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:28) at the amino acidscorresponding to amino acid positions 493 to 500 (VP1 numbering) of thenative AAV1 capsid protein, wherein X¹ is any amino acid other than K;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than D; wherein X is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;wherein X⁷ is any amino acid other than S; and X⁸ is any amino acidother than N. In particular embodiments, X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ IDNO:29) can be PGGNATR (SEQ ID NO:30).

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:31) at theamino acids corresponding to amino acid positions 588 to 597 (VP1numbering) of the native AAV1 capsid protein, wherein X¹ is any aminoacid other than S; wherein X² is any amino acid other than T; wherein X³is any amino acid other than D; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than A; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than D; wherein X⁹ is any amino acid other thanV; and wherein X^(1′) is any amino acid other than H. In particularembodiments, X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:31) can be: (a)TADHDTKGVL (SEQ ID NO:32); (b) VVDPDKKGVL (SEQ ID NO:33); (c) AKDTGPLNVM(SEQ ID NO:34); (d) QTDAKDNGVQ (SEQ ID NO:35); (e) DKDPWLNDVI (SEQ IDNO:36); (f) TRDGSTESVL (SEQ ID NO:37); (g) VIDPDQKGVL (SEQ ID NO:38); or(h) VNDMSNYMVH (SEQ ID NO:39).

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 709 to 710 (VP1 numbering) of the native AAV1 capsid protein,wherein X¹ is any amino acid other than A; and wherein X² is any aminoacid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:40) at the amino acidscorresponding to amino acid positions 716 to 722 (VP1 numbering) of thenative AAV1 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than N; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than L; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶ (SEQ ID NO:41) at the amino acidscorresponding to amino acid positions 262 to 267 (VP1 numbering) of thenative AAV2 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than Q; wherein X³ is any amino acidother than S; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than A; and wherein X⁶ is any amino acid other thanS.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:42) at theamino acids corresponding to amino acid positions 369 to 378 (VP1numbering) of the native AAV2 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanV; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:43) at the amino acidscorresponding to amino acid positions 455 to 458 (VP1 numbering) of thenative AAV2 capsid protein, wherein X¹ is any amino acid other than T;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than Q; and wherein X⁴ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:44) at the amino acidscorresponding to amino acid positions 492 to 498 (VP1 numbering) of thenative AAV2 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than A; wherein X³ is any amino acidother than D; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:45) at theamino acids corresponding to amino acid positions 587 to 596 (VP1numbering) of the native AAV2 capsid protein, wherein X¹ is any aminoacid other than N; wherein X² is any amino acid other than R; wherein X³is any amino acid other than Q; wherein X⁴ is any amino acid other thanA; wherein X⁵ is any amino acid other than A; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than A; wherein X⁸is any amino acid other than D; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 708 to 709 (VP1 numbering) of the native AAV12 capsid protein,wherein X¹ is any amino acid other than V; and wherein X² is any aminoacid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:46) at the amino acidscorresponding to amino acid positions 715 to 721 (VP1 numbering) of thenative AAV2 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than N; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than V; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶ (SEQ ID NO:47) at the amino acidscorresponding to amino acid positions 262 to 267 (VP1 numbering) of thenative AAV3 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than Q; wherein X³ is any amino acidother than S; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than A; and wherein X⁶ is any amino acid other thanS.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:48) at theamino acids corresponding to amino acid positions 369 to 378 (VP1numbering) of the native AAV3 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanV; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:49) at the amino acidscorresponding to amino acid positions 456 to 459 (VP1 numbering) of thenative AAV3 capsid protein, wherein X¹ is any amino acid other than T;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than Q; and wherein X⁴ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:50) at the amino acidscorresponding to amino acid positions 493 to 499 (VP1 numbering) of thenative AAV3 capsid protein, wherein X¹ is any amino acid other than A;wherein X² is any amino acid other than N; wherein X is any amino acidother than D; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-6-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:57) at the aminoacids corresponding to amino acid positions 588 to 597 (VP1 numbering)of the native AAV3 capsid protein, wherein X¹ is any amino acid otherthan N; wherein X² is any amino acid other than T; wherein X³ is anyamino acid other than A; wherein X⁴ is any amino acid other than P;wherein X⁵ is any amino acid other than T; wherein X⁶ is any amino acidother than T; wherein X⁷ is any amino acid other than G; wherein X⁸ isany amino acid other than T; wherein X⁹ is any amino acid other than V;and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 709 to 710 (VP1 numbering) of the native AAV3 capsid protein,wherein X¹ is any amino acid other than V; and wherein X² is any aminoacid other than N.

An adeno-associated virus (AAV) capsid protein, wherein the capsidprotein comprises a substitution at all positions or in any combinationof fewer than all positions, resulting in the amino acid sequence:X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:52) at the amino acids corresponding toamino acid positions 716 to 722 (VP1 numbering) of the native AAV3capsid protein, wherein X¹ is any amino acid other than D; wherein X² isany amino acid other than T; wherein X³ is any amino acid other than N;wherein X⁴ is any amino acid other than G; wherein X⁵ is any amino acidother than V; wherein X⁶ is any amino acid other than Y; and wherein X⁷is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:53) at the amino acidscorresponding to amino acid positions 253 to 260 (VP1 numbering) of thenative AAV4 capsid protein, wherein X¹ is any amino acid other than R;wherein X² is any amino acid other than L; wherein X³ is any amino acidother than G; wherein X⁴ is any amino acid other than E; wherein X⁵ isany amino acid other than S; wherein X⁶ is any amino acid other than L;wherein X⁷ is any amino acid other than Q; and wherein X⁸ is any aminoacid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:54) at theamino acids corresponding to amino acid positions 360 to 369 (VP1numbering) of the native AAV4 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanV; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY: and wherein X¹⁰ is any amino acid other than C.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:55) at the amino acidscorresponding to amino acid positions 450 to 453 (VP1 numbering) of thenative AAV4 capsid protein, wherein X¹ is any amino acid other than A;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than T; and wherein X⁴ is any amino acid other than A.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO:56) atthe amino acids corresponding to amino acid positions 487 to 498 (VP1numbering) of the native AAV4 capsid protein, wherein X¹ is any aminoacid other than A; wherein X² is any amino acid other than N; wherein X³is any amino acid other than Q; wherein X⁴ is any amino acid other thanN; wherein X⁵ is any amino acid other than Y; wherein X⁶ is any aminoacid other than K; wherein X⁷ is any amino acid other than I; wherein X⁸is any amino acid other than P; wherein X⁹ is any amino acid other thanA; wherein X¹⁰ is any amino acid other than T; wherein X¹¹ is any aminoacid other than G; and wherein X¹² is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:57) at theamino acids corresponding to amino acid positions 586 to 595 (VP1numbering) of the native AAV4 capsid protein, wherein X¹ is any aminoacid other than S; wherein X² is any amino acid other than N; wherein X³is any amino acid other than L; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than T; wherein X⁶ is any aminoacid other than V; wherein X⁷ is any amino acid other than D; wherein X⁸is any amino acid other than R; wherein X⁹ is any amino acid other thanL; and wherein X¹⁰ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 707 to 708 (VP1 numbering) of the native AAV4 capsid protein,wherein X¹ is any amino acid other than N; and wherein X² is any aminoacid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:58) at the amino acidscorresponding to amino acid positions 714 to 720 (VP1 numbering) of thenative AAV4 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than A; wherein X³ is any amino acidother than A; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than K; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:59) at theamino acids corresponding to amino acid positions 249 to 258 (VP1numbering) of the native AAV5 capsid protein, wherein X¹ is any aminoacid other than E; wherein X² is any amino acid other than I; wherein X³is any amino acid other than K; wherein X⁴ is any amino acid other thanS; wherein X⁵ is any amino acid other than G; wherein X⁶ is any aminoacid other than S; wherein X⁷ is any amino acid other than V; wherein X⁸is any amino acid other than D; wherein X⁹ is any amino acid other thanG; and wherein X¹⁰ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:60) at theamino acids corresponding to amino acid positions 360 to 369 (VP1numbering) of the native AAV5 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than T; wherein X⁴ is any amino acid other thanL; wherein X⁵ is any amino acid other than P wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than A.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:61) at the amino acidscorresponding to amino acid positions 443 to 446 (VP1 numbering) of thenative AAV5 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than G; and wherein X⁴ is any amino acid other than G.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:62) at the amino acidscorresponding to amino acid positions 479 to 485 (VP1 numbering) of thenative AAV5 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than V; wherein X⁴ is any amino acid other than N wherein X⁵ isany amino acid other than R; wherein X⁶ is any amino acid other than A;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:63) at theamino acids corresponding to amino acid positions 577 to 586 (VP1numbering) of the native AAV5 capsid protein, wherein X¹ is any aminoacid other than T; wherein X² is any amino acid other than T; wherein X³is any amino acid other than A; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than A; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than T; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 697 to 698 (VP1 numbering) of the native AAV5 capsid protein,wherein X¹ is any amino acid other than Q; and wherein X² is any aminoacid other than F.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:64) at the amino acidscorresponding to amino acid positions 704 to 710 (VP1 numbering) of thenative AAV5 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than S; wherein X is any amino acidother than T; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than E; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than R.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:65) at the amino acidscorresponding to amino acid positions 262 to 268 (VP1 numbering) of thenative AAV6 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than A; wherein X³ is any amino acidother than S; wherein X⁴ is any amino acid other than T; wherein X⁵ isany amino acid other than G; wherein X⁶ is any amino acid other than A;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:66) at theamino acids corresponding to amino acid positions 370 to 379 (VP1numbering) of the native AAV6 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanI; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:67) at the amino acidscorresponding to amino acid positions 456 to 459 (VP1 numbering) of thenative AAV6 capsid protein, wherein X¹ is any amino acid other than A;wherein X² is any amino acid other than Q; wherein X³ is any amino acidother than N; and wherein X⁴ is any amino acid other than K.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:68) at the amino acidscorresponding to amino acid positions 493 to 499 (VP1 numbering) of thenative AAV6 capsid protein, wherein X¹ is any amino acid other than K;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than D; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:69) at theamino acids corresponding to amino acid positions 588 to 597 (VP1numbering) of the native AAV6 capsid protein, wherein X¹ is any aminoacid other than S; wherein X² is any amino acid other than T; wherein X³is any amino acid other than D; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than A; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than D; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than H.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 709 to 710 (VP1 numbering) of the native AAV6 capsid protein,wherein X¹ is any amino acid other than A; and wherein X² is any aminoacid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:70) at the amino acidscorresponding to amino acid positions 716 to 722 (VP1 numbering) of thenative AAV6 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than N; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than L; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:71) at the amino acidscorresponding to amino acid positions 263 to 269 (VP1 numbering) of thenative AAV7 capsid protein, wherein X¹ is any amino acid other than S;wherein X² is any amino acid other than E; wherein X³ is any amino acidother than T; wherein X⁴ is any amino acid other than A; wherein X⁵ isany amino acid other than G; wherein X⁶ is any amino acid other than S;and wherein X⁷ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:72) at theamino acids corresponding to amino acid positions 371 to 380 (VP1numbering) of the native AAV7 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanI; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:73) at the amino acidscorresponding to amino acid positions 458 to 461 (VP1 numbering) of thenative AAV7 capsid protein, wherein X¹ is any amino acid other than A;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than N; and wherein X⁴ is any amino acid other than R.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:74) at the amino acidscorresponding to amino acid positions 495 to 501 (VP1 numbering) of thenative AAV7 capsid protein, wherein X¹ is any amino acid other than L;wherein X² is any amino acid other than D; wherein X³ is any amino acidother than Q; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:75) at theamino acids corresponding to amino acid positions 589 to 598 (VP1numbering) of the native AAV7 capsid protein, wherein X¹ is any aminoacid other than N; wherein X² is any amino acid other than T; wherein X³is any amino acid other than A; wherein X⁴ is any amino acid other thanA; wherein X⁵ is any amino acid other than Q; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than Q; wherein X⁸is any amino acid other than V; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution resulting in theamino acid sequence: X¹-X² at the amino acids corresponding to aminoacid positions 710 to 711 (VP1 numbering) of the native AAV7 capsidprotein, wherein X¹ is any amino acid other than T; and wherein X² isany amino acid other than G;

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:76) at the amino acidscorresponding to amino acid positions 717 to 723 (VP1 numbering) of thenative AAV7 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than S; wherein X³ is any amino acidother than Q; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than V; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:77) at the amino acidscorresponding to amino acid positions 263 to 270 (VP1 numbering) of thenative AAV8 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than T; wherein X⁴ is any amino acid other than S; wherein X⁵ isany amino acid other than G; wherein X⁶ is any amino acid other than G;wherein X⁷ is any amino acid other than A; and wherein X⁸ is any aminoacid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹⁻X¹⁰ (SEQ ID NO:78) at theamino acids corresponding to amino acid positions 372 to 381 (VP1numbering) of the native AAV8 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanI; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:79) at the amino acidscorresponding to amino acid positions 458 to 461 (VP1 numbering) of thenative AAV8 capsid protein, wherein X¹ is any amino acid other than A;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than T; and wherein X⁴ is any amino acid other than Q.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:80) at the amino acidscorresponding to amino acid positions 495 to 501 (VP1 numbering) of thenative AAV8 capsid protein, wherein X¹ is any amino acid other than T;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than Q; wherein X⁴ is any amino acid other than N wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹ (SEQ ID NO:81) at theamino acids corresponding to amino acid positions 590 to 600 (VP1numbering) of the native AAV8 capsid protein, wherein X¹ is any aminoacid other than N; wherein X² is any amino acid other than T; wherein X³is any amino acid other than A; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than Q; wherein X⁶ is any aminoacid other than I; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than T; wherein X⁹ is any amino acid other thanV; wherein X¹⁰ is any amino acid other than N; and wherein X¹¹ is anyamino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 711 to 712 (VP1 numbering) of the native AAV8 capsid protein,wherein X¹ is any amino acid other than T; and wherein X² is any aminoacid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:82) at the amino acidscorresponding to amino acid positions 718 to 724 (VP1 numbering) of thenative AAV8 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than E; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than V; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:83) at the amino acidscorresponding to amino acid positions 262 to 269 (VP1 numbering) of thenative AAV9 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than S; wherein X³ is any amino acidother than T; wherein X⁴ is any amino acid other than S; wherein X⁵ isany amino acid other than G; wherein X⁶ is any amino acid other than G;wherein X⁷ is any amino acid other than S; and wherein X⁸ is any aminoacid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:84) at theamino acids corresponding to amino acid positions 371 to 380 (VP1numbering) of the native AAV9 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanI; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:85) at the amino acidscorresponding to amino acid positions 456 to 459 (VP1 numbering) of thenative AAV9 capsid protein, wherein X¹ is any amino acid other than Q;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than Q; and wherein X⁴ is any amino acid other than Q.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:86) at the amino acidscorresponding to amino acid positions 493 to 499 (VP1 numbering) of thenative AAV9 capsid protein, wherein X¹ is any amino acid other than V;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than Q; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:87) at theamino acids corresponding to amino acid positions 588 to 597 (VP1numbering) of the native AAV9 capsid protein, wherein X¹ is any aminoacid other than Q; wherein X² is any amino acid other than A; wherein X³is any amino acid other than Q; wherein X⁴ is any amino acid other thanA; wherein X⁵ is any amino acid other than Q; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than W; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than Q.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 709 to 710 (VP1 numbering) of the native AAV9 capsid protein,wherein X¹ is any amino acid other than N; and wherein X² is any aminoacid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:88) at the amino acidscorresponding to amino acid positions 716 to 722 (VP1 numbering) of thenative AAV9 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than E; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than V; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:89) at the amino acidscorresponding to amino acid positions 263 to 270 (VP1 numbering) of thenative AAVrh10 capsid protein, wherein X¹ is any amino acid other thanN; wherein X² is any amino acid other than G; wherein X³ is any aminoacid other than T; wherein X⁴ is any amino acid other than S; wherein X⁵is any amino acid other than G; wherein X⁶ is any amino acid other thanG; wherein X⁷ is any amino acid other than S; and wherein X⁸ is anyamino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:90) at theamino acids corresponding to amino acid positions 372 to 381 (VP1numbering) of the native AAVrh10 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanI; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:91) at the amino acidscorresponding to amino acid positions 458 to 461 (VP1 numbering) of thenative AAVrh10 capsid protein, wherein X¹ is any amino acid other thanA; wherein X² is any amino acid other than G; wherein X³ is any aminoacid other than T; and wherein X⁴ is any amino acid other than Q.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:92) at the amino acidscorresponding to amino acid positions 495 to 501 (VP1 numbering) of thenative AAVrh10 capsid protein, wherein X¹ is any amino acid other thanL; wherein X² is any amino acid other than S; wherein X³ is any aminoacid other than Q; wherein X⁴ is any amino acid other than N; wherein X⁵is any amino acid other than N; wherein X⁶ is any amino acid other thanN; and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:93) at theamino acids corresponding to amino acid positions 590 to 599 (VP1numbering) of the native AAVrh10 capsid protein, wherein X¹ is any aminoacid other than N; wherein X² is any amino acid other than A; wherein X³is any amino acid other than A; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than I; wherein X⁶ is any aminoacid other than V; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than A; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 711 to 712 (VP1 numbering) of the native AAVrh10 capsidprotein, wherein X¹ is any amino acid other than T; and wherein X² isany amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:94) at the amino acidscorresponding to amino acid positions 718 to 724 (VP1 numbering) of thenative AAVrh10 capsid protein, wherein X¹ is any amino acid other thanN; wherein X² is any amino acid other than T; wherein X³ is any aminoacid other than D; wherein X⁴ is any amino acid other than G; wherein X⁵is any amino acid other than T; wherein X⁶ is any amino acid other thanY; and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:95) at the amino acidscorresponding to amino acid positions 262 to 269 (VP1 numbering) of thenative AAVrh8 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than T; wherein X is any amino acid other than S; wherein X⁵ isany amino acid other than G; wherein X⁶ is any amino acid other than G;wherein X⁷ is any amino acid other than S; and wherein X⁸ is any aminoacid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:96) at theamino acids corresponding to amino acid positions 371 to 380 (VP1numbering) of the native AAVrh8 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanV; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁵is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:97) at the amino acidscorresponding to amino acid positions 456 to 459 (VP1 numbering) of thenative AAVrh8 capsid protein, wherein X¹ is any amino acid other than G;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than T; and wherein X⁴ is any amino acid other than Q.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:98) at the amino acidscorresponding to amino acid positions 493 to 499 (VP1 numbering) of thenative AAVrh8 capsid protein, wherein X¹ is any amino acid other than T;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than Q; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:99) at theamino acids corresponding to amino acid positions 588 to 597 (VP1numbering) of the native AAVrh8 capsid protein, wherein X¹ is any aminoacid other than N; wherein X² is any amino acid other than T; wherein X³is any amino acid other than Q; wherein X⁴ is any amino acid other thanA; wherein X⁵ is any amino acid other than Q; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than L; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than H.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 709 to 710 (VP1 numbering) of the native AAVrh8 capsidprotein, wherein X¹ is any amino acid other than T; and wherein X² isany amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:100) at the amino acidscorresponding to amino acid positions 716 to 722 (VP1 numbering) of thenative AAVrh8 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than E; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than V; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:101) at the aminoacids corresponding to amino acid positions 263 to 270 (VP1 numbering)of the native AAV10 capsid protein, wherein X¹ is any amino acid otherthan N; wherein X² is any amino acid other than G; wherein X³ is anyamino acid other than T; wherein X⁴ is any amino acid other than S;wherein X⁵ is any amino acid other than G; wherein X⁶ is any amino acidother than G; wherein X⁷ is any amino acid other than S; and wherein X⁸is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:102) at theamino acids corresponding to amino acid positions 372 to 381 (VP1numbering) of the native AAV10 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanI; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than L.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:103) at the amino acidscorresponding to amino acid positions 458 to 461 (VP1 numbering) of thenative AAV10 capsid protein, wherein X¹ is any amino acid other than Q;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than T; and wherein X⁴ is any amino acid other than Q.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:104) at the amino acidscorresponding to amino acid positions 495 to 501 (VP1 numbering) of thenative AAV10 capsid protein, wherein X¹ is any amino acid other than L;wherein X² is any amino acid other than S; wherein X³ is any amino acidother than Q; wherein X⁴ is any amino acid other than N; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than N;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:105) at theamino acids corresponding to amino acid positions 590 to 599 (VP1numbering) of the native AAV10 capsid protein, wherein X¹ is any aminoacid other than N; wherein X² is any amino acid other than T; wherein X³is any amino acid other than G; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than I; wherein X⁶ is any aminoacid other than V; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than N; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 711 to 712 (VP1 numbering) of the native AAV10 capsid protein,wherein X¹ is any amino acid other than T; and wherein X² is any aminoacid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:106) at the amino acidscorresponding to amino acid positions 718 to 724 (VP1 numbering) of thenative AAV10 capsid protein, wherein X¹ is any amino acid other than N;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than E; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than T; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:107) at the aminoacids corresponding to amino acid positions 253 to 260 (VP1 numbering)of the native AAV11 capsid protein, wherein X¹ is any amino acid otherthan R; wherein X² is any amino acid other than L; wherein X³ is anyamino acid other than G; wherein X⁴ is any amino acid other than T;wherein X⁵ is any amino acid other than T; wherein X⁶ is any amino acidother than S; wherein X⁷ is any amino acid other than S; and wherein X⁸is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:108) at theamino acids corresponding to amino acid positions 360 to 369 (VP1numbering) of the native AAV11 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanV; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than C.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:109) at the amino acidscorresponding to amino acid positions 449 to 452 (VP1 numbering) of thenative AAV11 capsid protein, wherein X¹ is any amino acid other than Q;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than N; and wherein X⁴ is any amino acid other than A.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO:110) atthe amino acids corresponding to amino acid positions 486 to 497 (VP1numbering) of the native AAV11 capsid protein, wherein X¹ is any aminoacid other than A; wherein X² is any amino acid other than S; wherein X³is any amino acid other than Q; wherein X⁴ is any amino acid other thanN; wherein X⁵ is any amino acid other than Y; wherein X⁶ is any aminoacid other than K; wherein X⁷ is any amino acid other than I; wherein X⁸is any amino acid other than P; wherein X⁹ is any amino acid other thanA; wherein X¹⁰ is any amino acid other than S; wherein X¹¹ is any aminoacid other than G; and wherein X¹² is any amino acid other than G.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:111) at theamino acids corresponding to amino acid positions 585 to 594 (VP1numbering) of the native AAV11 capsid protein, wherein X¹ is any aminoacid other than T; wherein X² is any amino acid other than T; wherein X³is any amino acid other than A; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than I; wherein X⁶ is any aminoacid other than T; wherein X⁷ is any amino acid other than G; wherein X⁸is any amino acid other than N; wherein X⁹ is any amino acid other thanV; and wherein X¹⁰ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 706 to 707 (VP1 numbering) of the native AAV11 capsid protein,wherein X¹ is any amino acid other than S; and wherein X² is any aminoacid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:112) at the amino acidscorresponding to amino acid positions 713 to 719 (VP1 numbering) of thenative AAV1l capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than T; wherein X³ is any amino acidother than T; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than K; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:113) at the aminoacids corresponding to amino acid positions 262 to 269 (VP1 numbering)of the native AAV12 capsid protein, wherein X¹ is any amino acid otherthan R; wherein X² is any amino acid other than I; wherein X³ is anyamino acid other than G; wherein X⁴ is any amino acid other than T;wherein X⁵ is any amino acid other than T; wherein X⁶ is any amino acidother than A; wherein X⁷ is any amino acid other than N; and wherein X⁸is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:114) at theamino acids corresponding to amino acid positions 369 to 378 (VP1numbering) of the native AAV12 capsid protein, wherein X¹ is any aminoacid other than V; wherein X² is any amino acid other than F; wherein X³is any amino acid other than M; wherein X⁴ is any amino acid other thanV; wherein X⁵ is any amino acid other than P; wherein X⁶ is any aminoacid other than Q; wherein X⁷ is any amino acid other than Y; wherein X⁸is any amino acid other than G; wherein X⁹ is any amino acid other thanY; and wherein X¹⁰ is any amino acid other than C.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:115) at the amino acidscorresponding to amino acid positions 458 to 461 (VP1 numbering) of thenative AAV12 capsid protein, wherein X¹ is any amino acid other than Q;wherein X² is any amino acid other than G; wherein X³ is any amino acidother than T; and wherein X⁴ is any amino acid other than A.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO:116) atthe amino acids corresponding to amino acid positions 495 to 506 (VP1numbering) of the native AAV12 capsid protein, wherein X¹ is any aminoacid other than A; wherein X² is any amino acid other than N; wherein X³is any amino acid other than Q; wherein X⁴ is any amino acid other thanN; wherein X⁵ is any amino acid other than Y; wherein X⁶ is any aminoacid other than K; wherein X⁷ is any amino acid other than I; wherein X⁸is any amino acid other than P; wherein X⁹ is any amino acid other thanA; wherein X¹⁰ is any amino acid other than S; wherein X¹¹ is any aminoacid other than G; and wherein X¹² is any amino acid other than G.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:117) at theamino acids corresponding to amino acid positions 594 to 601 (VP1numbering) of the native AAV12 capsid protein, wherein X¹ is any aminoacid other than T; wherein X² is any amino acid other than T; wherein X³is any amino acid other than A; wherein X⁴ is any amino acid other thanP; wherein X⁵ is any amino acid other than H; wherein X⁶ is any aminoacid other than I; wherein X⁷ is any amino acid other than A; wherein X⁸is any amino acid other than N; wherein X⁹ is any amino acid other thanL; and wherein X¹⁰ is any amino acid other than D.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 715 to 716 (VP1 numbering) of the native AAV12 capsid protein,wherein X¹ is any amino acid other than N; and wherein X² is any aminoacid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:118) at the amino acidscorresponding to amino acid positions 722 to 728 (VP1 numbering) of thenative AAV12 capsid protein, wherein X¹ is any amino acid other than D;wherein X² is any amino acid other than N; wherein X³ is any amino acidother than A; wherein X⁴ is any amino acid other than G; wherein X⁵ isany amino acid other than N; wherein X⁶ is any amino acid other than Y;and wherein X⁷ is any amino acid other than H.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:119) at the aminoacids corresponding to amino acid positions 253 to 260 (VP1 numbering)of the native AAVrh32.33 capsid protein, wherein X¹ is any amino acidother than R; wherein X² is any amino acid other than L; wherein X³ isany amino acid other than G; wherein X⁴ is any amino acid other than T;wherein X⁵ is any amino acid other than T; wherein X⁶ is any amino acidother than S; wherein X⁷ is any amino acid other than N; and wherein X⁸is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:120) at theamino acids corresponding to amino acid positions 360 to 369 (VP1numbering) of the native AAVrh32.33 capsid protein, wherein X¹ is anyamino acid other than V; wherein X² is any amino acid other than F;wherein X³ is any amino acid other than M; wherein X⁴ is any amino acidother than V; wherein X⁵ is any amino acid other than P; wherein X⁶ isany amino acid other than Q; wherein X⁷ is any amino acid other than Y;wherein X⁸ is any amino acid other than G; wherein X⁹ is any amino acidother than Y; and wherein X¹⁰ is any amino acid other than C.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:121) at the amino acidscorresponding to amino acid positions 449 to 452 (VP1 numbering) of thenative AAVrh32.33 capsid protein, wherein X¹ is any amino acid otherthan Q; wherein X² is any amino acid other than G; wherein X³ is anyamino acid other than N; and wherein X⁴ is any amino acid other than A.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO:122) atthe amino acids corresponding to amino acid positions 486 to 497 (VP1numbering) of the native AAVrh32.33 capsid protein, wherein X¹ is anyamino acid other than A; wherein X² is any amino acid other than S;wherein X³ is any amino acid other than Q; wherein X⁴ is any amino acidother than N; wherein X⁵ is any amino acid other than Y; wherein X⁶ isany amino acid other than K; wherein X⁷ is any amino acid other than I;wherein X⁸ is any amino acid other than P; wherein X⁹ is any amino acidother than A; wherein X¹⁰ is any amino acid other than S; wherein X¹¹ isany amino acid other than G; and wherein X¹² is any amino acid otherthan G.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:123) at theamino acids corresponding to amino acid positions 585 to 594 (VP1numbering) of the native AAVrh32.33 capsid protein, wherein X¹ is anyamino acid other than T; wherein X² is any amino acid other than T;wherein X³ is any amino acid other than A; wherein X⁴ is any amino acidother than P; wherein X⁵ is any amino acid other than I; wherein X⁶ isany amino acid other than T; wherein X⁷ is any amino acid other than G;wherein X⁸ is any amino acid other than N; wherein X⁹ is any amino acidother than V; and wherein X¹⁰ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 706 to 707 (VP1 numbering) of the native AAVrh32.33 capsidprotein, wherein X¹ is any amino acid other than S; and wherein X² isany amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:124) at the amino acidscorresponding to amino acid positions 713 to 719 (VP1 numbering) of thenative AAVrh32.33 capsid protein, wherein X¹ is any amino acid otherthan D; wherein X² is any amino acid other than T; wherein X³ is anyamino acid other than T; wherein X⁴ is any amino acid other than G;wherein X⁵ is any amino acid other than K; wherein X⁶ is any amino acidother than Y; and wherein X⁷ is any amino acid other than T.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:125) at the aminoacids corresponding to amino acid positions 255 to 262 (VP1 numbering)of the native bovine AAV capsid protein, wherein X¹ is any amino acidother than R; wherein X² is any amino acid other than L; wherein X³ isany amino acid other than G; wherein X⁴ is any amino acid other than S;wherein X⁵ is any amino acid other than S; wherein X⁶ is any amino acidother than N; wherein X⁷ is any amino acid other than A; and wherein X⁸is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:126) at theamino acids corresponding to amino acid positions 362 to 371 (VP1numbering) of the native bovine AAV capsid protein, wherein X¹ is anyamino acid other than V; wherein X² is any amino acid other than F;wherein X³ is any amino acid other than M; wherein X⁴ is any amino acidother than V; wherein X⁵ is any amino acid other than P; wherein X⁶ isany amino acid other than Q; wherein X⁷ is any amino acid other than Y;wherein X⁸ is any amino acid other than G; wherein X⁹ is any amino acidother than Y; and wherein X¹⁰ is any amino acid other than C.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:127) at the amino acidscorresponding to amino acid positions 452 to 455 (VP1 numbering) of thenative bovine AAV capsid protein, wherein X¹ is any amino acid otherthan Q; wherein X² is any amino acid other than G; wherein X³ is anyamino acid other than N; and wherein X⁴ is any amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO:128) atthe amino acids corresponding to amino acid positions 489 to 500 (VP1numbering) of the native bovine AAV capsid protein, wherein X¹ is anyamino acid other than A; wherein X² is any amino acid other than S;wherein X³ is any amino acid other than Q; wherein X⁴ is any amino acidother than N; wherein X⁵ is any amino acid other than Y; wherein X⁶ isany amino acid other than K; wherein X⁷ is any amino acid other than I;wherein X⁸ is any amino acid other than P; wherein X⁹ is any amino acidother than Q; wherein X¹⁰ is any amino acid other than G; wherein X¹¹ isany amino acid other than R; and wherein X¹² is any amino acid otherthan N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:129) at theamino acids corresponding to amino acid positions 588 to 597 (VP1numbering) of the native bovine AAV capsid protein, wherein X¹ is anyamino acid other than T; wherein X² is any amino acid other than T;wherein X³ is any amino acid other than V; wherein X⁴ is any amino acidother than P; wherein X⁵ is any amino acid other than T; wherein X⁶ isany amino acid other than V; wherein X⁷ is any amino acid other than D;wherein X⁸ is any amino acid other than D; wherein X⁹ is any amino acidother than V; and wherein X¹⁰ is any amino acid other than D.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 709 to 710 (VP1 numbering) of the native bovine AAV capsidprotein, wherein X¹ is any amino acid other than D; and wherein X² isany amino acid other than S.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO: 130) at the amino acidscorresponding to amino acid positions 716 to 722 (VP1 numbering) of thenative bovine AAV capsid protein, wherein X¹ is any amino acid otherthan D; wherein X² is any amino acid other than N; wherein X³ is anyamino acid other than A; wherein X⁴ is any amino acid other than G;wherein X⁵ is any amino acid other than A; wherein X⁶ is any amino acidother than Y; and wherein X⁷ is any amino acid other than K.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:131) at the aminoacids corresponding to amino acid positions 265 to 272 (VP1 numbering)of the native avian AAV capsid protein, wherein X¹ is any amino acidother than R; wherein X² is any amino acid other than I; wherein X³ isany amino acid other than Q; wherein X⁴ is any amino acid other than G;wherein X⁵ is any amino acid other than P; wherein X⁶ is any amino acidother than S; wherein X⁷ is any amino acid other than G; and wherein X⁸is any amino acid other than G.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:132) at theamino acids corresponding to amino acid positions 375 to 384 (VP1numbering) of the native avian AAV capsid protein, wherein X¹ is anyamino acid other than I; wherein X² is any amino acid other than Y;wherein X³ is any amino acid other than T; wherein X⁴ is any amino acidother than I; wherein X⁵ is any amino acid other than P; wherein X⁶ isany amino acid other than Q; wherein X⁷ is any amino acid other than Y;wherein X⁸ is any amino acid other than G; wherein X⁹ is any amino acidother than Y; and wherein X¹⁰ is any amino acid other than C.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴ (SEQ ID NO:133) at the amino acidscorresponding to amino acid positions 459 to 462 (VP1 numbering) of thenative avian AAV capsid protein, wherein X¹ is any amino acid other thanS; wherein X² is any amino acid other than S; wherein X³ is any aminoacid other than G; and wherein X⁴ is any amino acid other than R.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO:134) atthe amino acids corresponding to amino acid positions 496 to 507 (VP1numbering) of the native avian AAV capsid protein, wherein X¹ is anyamino acid other than A; wherein X² is any amino acid other than S;wherein X³ is any amino acid other than N; wherein X⁴ is any amino acidother than I; wherein X⁵ is any amino acid other than T; wherein X⁶ isany amino acid other than K; wherein X⁷ is any amino acid other than N;wherein X⁸ is any amino acid other than N; wherein X⁹ is any amino acidother than V; wherein X¹⁰ is any amino acid other than F; wherein X¹¹ isany amino acid other than S; and wherein X¹² is any amino acid otherthan V.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:135) at theamino acids corresponding to amino acid positions 595 to 604 (VP1numbering) of the native avian AAV capsid protein, wherein X¹ is anyamino acid other than V; wherein X² is any amino acid other than T;wherein X³ is any amino acid other than P; wherein X⁴ is any amino acidother than G; wherein X⁵ is any amino acid other than T; wherein X⁶ isany amino acid other than R; wherein X⁷ is any amino acid other than A;wherein X⁸ is any amino acid other than A; wherein X⁹ is any amino acidother than V; and wherein X¹⁰ is any amino acid other than N.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X² at the amino acids corresponding to amino acidpositions 716 to 717 (VP1 numbering) of the native avian AAV capsidprotein, wherein X¹ is any amino acid other than A; and wherein X² isany amino acid other than D.

An adeno-associated virus (AAV) capsid protein is also provided herein,wherein the capsid protein comprises a substitution at all positions orin any combination of fewer than all positions, resulting in the aminoacid sequence: X¹-X²-X³-X⁴-X⁵-X⁶-X⁷ (SEQ ID NO:136) at the amino acidscorresponding to amino acid positions 723 to 729 (VP1 numbering) of thenative avian AAV capsid protein, wherein X¹ is any amino acid other thanS; wherein X² is any amino acid other than D; wherein X³ is any aminoacid other than T; wherein X⁴ is any amino acid other than G; wherein X⁵is any amino acid other than S; wherein X⁶ is any amino acid other thanY; and wherein X⁷ is any amino acid other than S.

In embodiments wherein any amino acid residue identified as X¹ throughX¹⁰ is not substituted, the amino acid residue at the unsubstitutedposition is the wild type amino acid residue of the reference amino acidsequence.

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 488R, 450Q, 453S, 454G, 455S, 456A, 457Q and/or500N of SEQ ID NO:1 (AAV1 capsid protein; VP1 numbering) in anycombination.

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 256L, 258K, 259Q, 261S, 263A, 264S, 265T, 266G,272H, 385S, 386Q, 547S, 709A, 710N, 716D, 717N, 718N, 720L and/or 722Tof SEQ ID NO:1 (AAV1 capsid protein; VP1 numbering) in any combination.

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 244N, 246Q, 248R, 249E, 2501, 251K, 252S, 253G,254S, 255V, 256D, 263Y, 377E, 378N, 453L, 456R, 532Q, 533P, 535N, 536P,537G, 538T, 539T, 540A, 541T, 542Y, 543L, 546N, 653V, 654P, 656S, 697Q,698F, 704D, 705S, 706T, 707G, 708E, 709Y and/or 710R of SEQ ID NO:5(AAV5 capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 248R, 316V, 317Q, 318D, 319S, 443N, 530N, 531S,532Q 533P, 534A, 535N, 540A, 541T, 542Y, 543L, 545G, 546N, 697Q, 704D,706T, 708E, 709Y and/or 710R of SEQ ID NO:5 (AAV5 capsid protein; VP1numbering) in any combination.

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 264S, 266G, 269N, 272H, 457Q, 588S and/or 589Tof SEQ ID NO:6 (AAV6 capsid protein; VP1 numbering) in any combination.

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 457T, 459N, 496G, 499N, 500N, 589Q, 590N and/or592A of SEQ ID NO:8 (AAV8 capsid protein; VP1 numbering) in anycombination.

An AAV capsid protein is also provided herein, comprising an amino acidsubstitution at residues 451, 452N, 453G, 454S, 455G, 456Q, 457N and/or458Q of SEQ ID NO:9 (AAV9 capsid protein; VP1 numbering) in anycombination.

An AAV capsid protein is also provided herein, comprising a S472Rsubstitution in the amino acid sequence of SEQ ID NO:1 (AAV1 capsidprotein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising a V473Dsubstitution in the amino acid sequence of SEQ ID NO:1 (AAV1 capsidprotein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising a N500Esubstitution in the amino acid sequence of SEQ ID NO:1 (AAV1 capsidprotein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising an A456T,Q457T, N458Q and K459S substation in the amino acid sequence of SEQ IDNO:1 (AAV1 capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising a T492S andK493A substitution in the amino acid sequence of SEQ ID NO:1 (AAV1capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising a S586R,S587G, S588N and T589R substitution in the amino acid sequence of SEQ IDNO:1 (AAV1 capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising an A456T,Q457T, N458Q, K459S, T492S and K493A substitution in the amino acidsequence of SEQ ID NO:1 (AAV1 capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising an A456T,Q457T, N458Q, K459S, S586R, S587G, S588N and T589R substitution in theamino acid sequence of SEQ ID NO:1 (AAV1 capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising a T492S,K493A, S586R, S587G, S588N and T589R substitution in the amino acidsequence of SEQ ID NO:1 (AAV1 capsid protein; VP1 numbering).

An AAV capsid protein is also provided herein, comprising an A456T,Q457T, N458Q, K459S, T492S, K493A, S586R, S587G, S588N and T589Rsubstitution in the amino acid sequence of SEQ ID NO:1 (AAV1 capsidprotein; VP1 numbering).

The present invention further provides an AAV capsid protein comprisingone or more amino acid substitutions of this invention, in anycombination. For example, an AAV capsid protein of any given serotypedescribed herein can comprise substitutions at the amino acid residuesidentified for CAM1, CAM3, CAM4-1, CAM4-2, CAM5, CAM6, CAM7, CAM8,CAM9-1 and/or CAM9-2 (listed in Table 5), singly or in any combination.As a further example, an AAV capsid of a first serotype can compriseamino acid substitutions that introduce residues that define a CAMregion of a different AAV serotype, which can be a second, third, fourthAAV serotype, etc. The CAM regions of different AAV serotypes can bepresent on a first AAV serotype in any combination. This cumulativeapproach generates novel AAVe strains, which present variable antigenicsurface topologies that would evade neutralizing antibodies. As aparticular, nonlimiting example, an AAV1 serotype capsid protein cancomprise an endogenous or mutated CAM1 region from a different secondAAV serotype and an endogenous or mutated CAM3 region of a differentthird serotype and an endogenous or mutated CAM4 region of a differentfourth serotype, etc., in any combination, as would be recognized by oneof ordinary skill in the art.

In particular embodiments, the modified virus capsid proteins of theinvention are not limited to AAV capsid proteins in which amino acidsfrom one AAV capsid protein are substituted into another AAV capsidprotein, and the substituted and/or inserted amino acids can be from anysource, and can further be naturally occurring or partially orcompletely synthetic.

As described herein, the nucleic acid and amino acid sequences of thecapsid proteins from a number of AAV are known in the art. Thus, theamino acids “corresponding” to amino acid positions of the native AAVcapsid protein can be readily determined for any other AAV (e.g., byusing sequence alignments).

The invention contemplates that the modified capsid proteins of theinvention can be produced by modifying the capsid protein of any AAV nowknown or later discovered. Further, the AAV capsid protein that is to bemodified can be a naturally occurring AAV capsid protein (e.g., an AAV2,AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10 or AAV11 capsid protein orany of the AAV shown in Table 1) but is not so limited. Those skilled inthe art will understand that a variety of manipulations to the AAVcapsid proteins are known in the art and the invention is not limited tomodifications of naturally occurring AAV capsid proteins. For example,the capsid protein to be modified may already have alterations ascompared with naturally occurring AAV (e.g., is derived from a naturallyoccurring AAV capsid protein, e.g., AAV2, AAV3a, AAV3b, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or any other AAV now knownor later discovered). Such AAV capsid proteins are also within the scopeof the present invention.

Thus, in particular embodiments, the AAV capsid protein to be modifiedcan be derived from a naturally occurring AAV but further comprise oneor more foreign sequences (e.g., that are exogenous to the native virus)that are inserted and/or substituted into the capsid protein and/or hasbeen altered by deletion of one or more amino acids.

Accordingly, when referring herein to a specific AAV capsid protein(e.g., an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11capsid protein or a capsid protein from any of the AAV shown in Table 1,etc.), it is intended to encompass the native capsid protein as well ascapsid proteins that have alterations other than the modifications ofthe invention. Such alterations include substitutions, insertions and/ordeletions. In particular embodiments, the capsid protein comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, lessthan 20, less than 30, less than 40 less than 50, less than 60, or lessthan 70 amino acids inserted therein (other than the insertions of thepresent invention) as compared with the native AAV capsid proteinsequence. In embodiments of the invention, the capsid protein comprises1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20,less than 20, less than 30, less than 40 less than 50, less than 60, orless than 70 amino acid substitutions (other than the amino acidsubstitutions according to the present invention) as compared with thenative AAV capsid protein sequence. In embodiments of the invention, thecapsid protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30,less than 40 less than 50, less than 60, or less than 70 amino acids(other than the amino acid deletions of the invention) as compared withthe native AAV capsid protein sequence.

Thus, for example, the term “AAV2 capsid protein” includes AAV capsidproteins having the native AAV2 capsid protein sequence (see GenBankAccession No. AAC03780) as well as those comprising substitutions,insertions and/or deletions (as described in the preceding paragraph) inthe native AAV2 capsid protein sequence.

In particular embodiments, the AAV capsid protein has the native AAVcapsid protein sequence or has an amino acid sequence that is at leastabout 90%, 95%, 97%, 98% or 99% similar or identical to a native AAVcapsid protein sequence. For example, in particular embodiments, an“AAV2” capsid protein encompasses the native AAV2 capsid proteinsequence as well as sequences that are at least about 90%, 95%, 97%, 98%or 99% similar or identical to the native AAV2 capsid protein sequence.

Methods of determining sequence similarity or identity between two ormore amino acid sequences are known in the art. Sequence similarity oridentity may be determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequenceidentity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman,Proc. Natl. Acad Sci. USA 85, 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.), the Best Fit sequence program describedby Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or byinspection.

Another suitable algorithm is the BLAST algorithm, described in Altschulet al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc.Natl. Acad Sci. USA 90, 5873-5787 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Methods in Enzymology, 266, 460-480 (1996);blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, which are optionally set to the default values. Theparameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

Further, an additional useful algorithm is gapped BLAST as reported byAltschul et al., (1997) Nucleic Acids Res. 25, 3389-3402.

The invention also provides a virus capsid comprising, consistingessentially of, or consisting of the modified AAV capsid protein of theinvention. In particular embodiments, the virus capsid is a parvoviruscapsid, which may further be an autonomous parvovirus capsid or adependovirus capsid. Optionally, the virus capsid is an AAV capsid. Inparticular embodiments, the AAV capsid is an AAV1, AAV2, AAV3a, AAV3b,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8,AAVrh10, AAVrh32.33, bovine AAV capsid, avian AAV capsid or any otherAAV now known or later identified. A nonlimiting list of AAV serotypesis shown in Table 1 an AAV capsid of this invention can be any AAVserotype listed in Table 1 or derived from any of the foregoing by oneor more insertions, substitutions and/or deletions.

The modified virus capsids can be used as “capsid vehicles,” as has beendescribed, for example, in U.S. Pat. No. 5,863,541. Molecules that canbe packaged by the modified virus capsid and transferred into a cellinclude heterologous DNA, RNA, polypeptides, small organic molecules,metals, or combinations of the same.

Heterologous molecules are defined as those that are not naturally foundin an AAV infection, e.g., those not encoded by a wild-type AAV genome.Further, therapeutically useful molecules can be associated with theoutside of the chimeric virus capsid for transfer of the molecules intohost target cells. Such associated molecules can include DNA, RNA, smallorganic molecules, metals, carbohydrates, lipids and/or polypeptides. Inone embodiment of the invention the therapeutically useful molecule iscovalently linked (i.e., conjugated or chemically coupled) to the capsidproteins. Methods of covalently linking molecules are known by thoseskilled in the art.

The modified virus capsids of the invention also find use in raisingantibodies against the novel capsid structures. As a furtheralternative, an exogenous amino acid sequence may be inserted into themodified virus capsid for antigen presentation to a cell, e.g., foradministration to a subject to produce an immune response to theexogenous amino acid sequence.

In other embodiments, the virus capsids can be administered to blockcertain cellular sites prior to and/or concurrently with (e.g., withinminutes or hours of each other) administration of a virus vectordelivering a nucleic acid encoding a polypeptide or functional RNA ofinterest. For example, the inventive capsids can be delivered to blockcellular receptors on liver cells and a delivery vector can beadministered subsequently or concurrently, which may reduce transductionof liver cells, and enhance transduction of other targets (e.g.,skeletal, cardiac and/or diaphragm muscle).

According to representative embodiments, modified virus capsids can beadministered to a subject prior to and/or concurrently with a modifiedvirus vector according to the present invention. Further, the inventionprovides compositions and pharmaceutical formulations comprising theinventive modified virus capsids; optionally, the composition alsocomprises a modified virus vector of the invention.

The invention also provides nucleic acids (optionally, isolated nucleicacids) encoding the modified virus capsids and capsid proteins of theinvention. Further provided are vectors comprising the nucleic acids,and cells (in vivo or in culture) comprising the nucleic acids and/orvectors of the invention. As one example, the present invention providesa virus vector comprising: (a) a modified AAV capsid of this invention;and (b) a nucleic acid comprising at least one terminal repeat sequence,wherein the nucleic acid is encapsidated by the AAV capsid.

Other suitable vectors include without limitation viral vectors (e.g.,adenovirus, AAV, herpesvirus, vaccinia, poxviruses, baculoviruses, andthe like), plasmids, phage, YACs, BACs, and the like. Such nucleicacids, vectors and cells can be used, for example, as reagents (e.g.,helper packaging constructs or packaging cells) for the production ofmodified virus capsids or virus vectors as described herein.

Virus capsids according to the invention can be produced using anymethod known in the art, e.g., by expression from a baculovirus (Brownet al., (1994) Virology 198:477-488).

The modifications to the AAV capsid protein according to the presentinvention are “selective” modifications. This approach is in contrast toprevious work with whole subunit or large domain swaps between AAVserotypes (see, e.g., international patent publication WO 00/28004 andHauck et al., (2003) J. Virology 77:2768-2774). In particularembodiments, a “selective” modification results in the insertion and/orsubstitution and/or deletion of less than about 20, 18, 15, 12, 10, 9,8, 7, 6, 5, 4 or 3 contiguous amino acids.

The modified capsid proteins and capsids of the invention can furthercomprise any other modification, now known or later identified.

For example, the AAV capsid proteins and virus capsids of the inventioncan be chimeric in that they can comprise all or a portion of a capsidsubunit from another virus, optionally another parvovirus or AAV, e.g.,as described in international patent publication WO 00/28004.

In some embodiments of this invention, the virus capsid can be atargeted virus capsid, comprising a targeting sequence (e.g.,substituted or inserted in the viral capsid) that directs the viruscapsid to interact with cell-surface molecules present on desired targettissue(s) (see, e.g., international patent publication WO 00/28004 andHauck et al., (2003) J. Virology 77:2768-2774); Shi et al., Human GeneTherapy 17:353-361 (2006) [describing insertion of the integrin receptorbinding motif RGD at positions 520 and/or 584 of the AAV capsidsubunit]; and U.S. Pat. No. 7,314,912 [describing insertion of the P1peptide containing an RGD motif following amino acid positions 447, 534,573 and 587 of the AAV2 capsid subunit]). Other positions within the AAVcapsid subunit that tolerate insertions are known in the art (e.g.,positions 449 and 588 described by Grifman et al., Molecular Therapy3:964-975 (2001)).

For example, a virus capsid of this invention may have relativelyinefficient tropism toward certain target tissues of interest (e.g.,liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach,intestines, skin, endothelial cells, and/or lungs). A targeting sequencecan advantageously be incorporated into these low-transduction vectorsto thereby confer to the virus capsid a desired tropism and, optionally,selective tropism for particular tissue(s). AAV capsid proteins, capsidsand vectors comprising targeting sequences are described, for example ininternational patent publication WO 00/28004. As another example, one ormore non-naturally occurring amino acids as described by Wang et al.,Annu Rev Biophys Biomol Struct. 35:225-49 (2006)) can be incorporatedinto an AAV capsid subunit of this invention at an orthogonal site as ameans of redirecting a low-transduction vector to desired targettissue(s). These unnatural amino acids can advantageously be used tochemically link molecules of interest to the AAV capsid proteinincluding without limitation: glycans (mannose-dendritic celltargeting); RGD, bombesin or a neuropeptide for targeted delivery tospecific cancer cell types; RNA aptamers or peptides selected from phagedisplay targeted to specific cell surface receptors such as growthfactor receptors, integrins, and the like. Methods of chemicallymodifying amino acids are known in the art (see, e.g., Greg T.Hermanson, Bioconjugate Techniques, 1^(st) edition, Academic Press,1996).

In some embodiments, the targeting sequence may be a virus capsidsequence (e.g., an autonomous parvovirus capsid sequence, AAV capsidsequence, or any other viral capsid sequence) that directs infection toa particular cell type(s).

As another nonlimiting example, a heparin binding domain (e.g., therespiratory syncytial virus heparin binding domain) may be inserted orsubstituted into a capsid subunit that does not typically bind HSreceptors (e.g., AAV4, AAV5) to confer heparin binding to the resultingmutant.

B19 infects primary erythroid progenitor cells using globoside as itsreceptor (Brown et al., (1993) Science 262:114). The structure of B19has been determined to 8 Å resolution (Agbandje-McKenna et al., (1994)Virology 203:106). The region of the B19 capsid that binds to globosidehas been mapped between amino acids 399-406 (Chapman et al., (1993)Virology 194:419), a looped out region between n3-barrel structures Eand F (Chipman et al., (1996) Proc. Nat. Acad Sci. USA 93:7502).Accordingly, the globoside receptor binding domain of the B19 capsid maybe substituted into an AAV capsid protein of this invention to target avirus capsid or virus vector comprising the same to erythroid cells.

In some embodiments, the exogenous targeting sequence may be any aminoacid sequence encoding a peptide that alters the tropism of a viruscapsid or virus vector comprising the modified AAV capsid protein. Inparticular embodiments, the targeting peptide or protein may benaturally occurring or, alternately, completely or partially synthetic.Exemplary targeting sequences include ligands and other peptides thatbind to cell surface receptors and glycoproteins, such as RGD peptidesequences, bradykinin, hormones, peptide growth factors (e.g., epidermalgrowth factor, nerve growth factor, fibroblast growth factor,platelet-derived growth factor, insulin-like growth factors I and II,etc.), cytokines, melanocyte stimulating hormone (e.g., α, β or γ),neuropeptides and endorphins, and the like, and fragments thereof thatretain the ability to target cells to their cognate receptors. Otherillustrative peptides and proteins include substance P, keratinocytegrowth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2,hen egg white lysozyme, erythropoietin, gonadoliberin, corticostatin,β-endorphin, leu-enkephalin, rimorphin, α-neo-enkephalin, angiotensin,pneumadin, vasoactive intestinal peptide, neurotensin, motilin, andfragments thereof as described above. As yet a further alternative, thebinding domain from a toxin (e.g., tetanus toxin or snake toxins, suchas α-bungarotoxin, and the like) can be substituted into the capsidprotein as a targeting sequence. In a yet further representativeembodiment, the AAV capsid protein can be modified by substitution of a“nonclassical” import/export signal peptide (e.g., fibroblast growthfactor-1 and -2, interleukin 1, HIV-1 Tat protein, herpes virus VP22protein, and the like) as described by Cleves (Current Biology 7:R318(1997)) into the AAV capsid protein. Also encompassed are peptide motifsthat direct uptake by specific cells, e.g., a FVFLP (SEQ ID NO:162)peptide motif triggers uptake by liver cells.

Phage display techniques, as well as other techniques known in the art,may be used to identify peptides that recognize any cell type ofinterest.

The targeting sequence may encode any peptide that targets to a cellsurface binding site, including receptors (e.g., protein, carbohydrate,glycoprotein or proteoglycan). Examples of cell surface binding sitesinclude, but are not limited to, heparan sulfate, chondroitin sulfate,and other glycosaminoglycans, sialic acid moieties found on mucins,glycoproteins, and gangliosides, MHC I glycoproteins, carbohydratecomponents found on membrane glycoproteins, including, mannose,N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, and thelike.

In particular embodiments, a heparan sulfate (HS) or heparin bindingdomain is substituted into the virus capsid (for example, in an AAVcapsid that otherwise does not bind to HS or heparin). It is known inthe art that HS/heparin binding is mediated by a “basic patch” that isrich in arginines and/or lysines. In exemplary embodiments, a sequencefollowing the motif BXXB (SEQ ID NO:163), where “B” is a basic residueand X is neutral and/or hydrophobic can be employed. As a nonlimitingexample, BXXB can be RGNR (SEQ ID NO:164). As another nonlimitingexample, BXXB is substituted for amino acid positions 262 through 265 inthe native AAV2 capsid protein or at the corresponding position(s) inthe capsid protein of another AAV serotype.

Other nonlimiting examples of suitable targeting sequences include thepeptides targeting coronary artery endothelial cells identified by Mlleret al., Nature Biotechnology 21:1040-1046 (2003) (consensus sequencesNSVRDL(G/S) (SEQ ID NO:165), PRSVTVP (SEQ ID NO:166), NSVSSX(S/A) (SEQID NO:167); tumor-targeting peptides as described by Grifman et al.,Molecular Therapy 3:964-975 (2001) (e.g., NGR, NGRAHA, SEQ ID NO:168);lung or brain targeting sequences as described by Work et al., MolecularTherapy 13:683-693 (2006) (QPEHSST; SEQ ID NO:169, VNTANST; SEQ IDNO:170, HGPMQKS; SEQ ID NO:171, PHKPPLA; SEQ ID NO:172, IKNNEMW; SEQ IDNO:173, RNLDTPM; SEQ ID NO:174, VDSHRQS; SEQ ID NO:175, YDSKTKT; SEQ IDNO:176, SQLPHQK; SEQ ID NO:177, STMQQNT; SEQ ID NO:178, TERYMTQ; SEQ IDNO:179, QPEHSST; SEQ ID NO:180, DASLSTS; SEQ ID NO:181, DLPNKKT; SEQ IDNO:182, DLTAARL; SEQ ID NO:183, EPHQFNY; SEQ ID NO:184, EPQSNHT; SEQ IDNO:185, MSSWPSQ; SEQ ID NO:186, NPKHNAT; SEQ ID NO:187, PDGMRTT; SEQ IDNO:188, PNNNKTT; SEQ ID NO:189, QSTTHDS; SEQ ID NO:190, TGSKQKQ; SEQ IDNO:191, SLKHQAL; SEQ ID NO:192 and SPIDGEQ; SEQ ID NO:193); vasculartargeting sequences described by Hajitou et al., TCM 16:80-88 (2006)(WIFPWIQL; SEQ ID NO:194, CDCRGDCFC; SEQ ID NO:195, CNGRC; SEQ IDNO:196, CPRECES; SEQ ID NO:197, GSL, CTTHWGFTLC; SEQ ID NO:198,CGRRAGGSC; SEQ ID NO:199, CKGGRAKDC; SEQ ID NO:200, and CVPELGHEC; SEQID NO:201); targeting peptides as described by Koivunen et al., J. Nucl.Med 40:883-888 (1999) (CRRETAWAK; SEQ ID NO:202, KGD, VSWFSHRYSPFAVS;SEQ ID NO:203, GYRDGYAGPILYN; SEQ ID NO:204, XXXY*XXX (SEQ ID NO:205)[where Y* is phospho-Tyr], Y*E/MNW; SEQ ID NO:206, RPLPPLP; SEQ IDNO:207, APPLPPR; SEQ ID NO:208, DVFYPYPYASGS; SEQ ID NO:209, MYWYPY; SEQID NO:210, DITWDQLWDLMK; SEQ ID NO:211, CWDD(G/L)WLC; SEQ ID NO:212,EWCEYLGGYLRCYA; SEQ ID NO:213, YXCXXGPXTWXCXP; SEQ ID NO:214,IEGPTLRQWLAARA; SEQ ID NO:215, LWXX(Y/W/F/H); SEQ ID NO:216, XFXXYLW;SEQ ID NO:217, SSIISHFRWGLCD; SEQ ID NO:218, MSRPACPPNDKYE; SEQ IDNO:219, CLRSGRGC; SEQ ID NO:220, CHWMFSPWC; SEQ ID NO:221, WXXF; SEQ IDNO:222, CSSRLDAC; SEQ ID NO:223, CLPVASC; SEQ ID NO:224, CGFECVRQCPERC;SEQ ID NO:225, CVALCREACGEGC; SEQ ID NO:226, SWCEPGWCR; SEQ ID NO:227,YSGKWGW; SEQ ID NO:228, GLSGGRS; SEQ ID NO:229, LMLPRAD; SEQ ID NO:230,CSCFRDVCC; SEQ ID NO:231, CRDVVSVIC; SEQ ID NO:232, CNGRC; SEQ IDNO:233, and GSL); and tumor targeting peptides as described by Newton &Deutscher, Phage Peptide Display in Handbook of ExperimentalPharmacology, pages 145-163, Springer-Verlag, Berlin (2008) (MARSGL; SEQID NO:234, MARAKE; SEQ ID NO:235, MSRTMS; SEQ ID NO:236, KCCYSL; SEQ IDNO:237, WRR, WKR, WVR, WVK, WIK, WTR, WVL, WLL, WRT, WRG, WVS, WVA,MYWGDSHWLQYWYE; SEQ ID NO:238, MQLPLAT; SEQ ID NO:239, EWLS; SEQ IDNO:240, SNEW; SEQ ID NO:241, TNYL; SEQ ID NO:242, WIFPWIQL; SEQ IDNO:243, WDLAWMFRLPVG; SEQ ID NO:244, CTVALPGGYVRVC; SEQ ID NO:245,CVPELGHEC; SEQ ID NO:246, CGRRAGGSC; SEQ ID NO:247, CVAYCIEHHCWTC; SEQID NO:248, CVFAHNYDYLVC; SEQ ID NO:249, and CVFTSNYAFC; SEQ ID NO:250,VHSPNKK; SEQ ID NO:251, CDCRGDCFC; SEQ ID NO:252, CRGDGWC; SEQ IDNO:253, XRGCDX; SEQ ID NO:254, PXX(S/T); SEQ ID NO:255, CTTHWGFTLC; SEQID NO:256, SGKGPRQITAL; SEQ ID NO:257, A(A/Q)(N/AX)(IY)(TN/V/M/RXR/K);SEQ ID NO:258, VYMSPF; SEQ ID NO:259, MQLPLAT; SEQ ID NO:260, ATWLPPR;SEQ ID NO:261, HTMYYHHYQHHL; SEQ ID NO:262, SEVGCRAGPLQWLCEKYFG; SEQ IDNO:263, CGLLPVGRPDRNVWRWLC; SEQ ID NO:264, CKGQCDRFKGLPWEC; SEQ IDNO:265, SGRSA; SEQ ID NO:266, WGFP; SEQ ID NO:267, LWXXAr [Ar=Y, W, F,H); SEQ ID NO:216, XFXXYLW; SEQ ID NO:268, AEPMPHSLNFSQYLWYT; SEQ IDNO:269, WAY(W/F)SP; SEQ ID NO:270, IELLQAR; SEQ ID NO:271, DITWDQLWDLMK;SEQ ID NO:272, AYTKCSRQWRTCMTTH; SEQ ID NO:273, PQNSKIPGPTFLDPH; SEQ IDNO:274, SMEPALPDWWWKMFK; SEQ ID NO:275, ANTPCGPYTHDCPVKR; SEQ ID NO:276,TACHQHVRMVRP; SEQ ID NO:277, VPWMEPAYQRFL; SEQ ID NO:278, DPRATPGS; SEQID NO:279, FRPNRAQDYNTN; SEQ ID NO:280, CTKNSYLMC; SEQ ID NO:281,C(R/Q)L/RT(G/N)XXG(A/V)GC; SEQ ID NO:282, CPIEDRPMC; SEQ ID NO:283,HEWSYLAPYPWF; SEQ ID NO:284, MCPKHPLGC; SEQ ID NO:285, RMWPSSTVNLSAGRR;SEQ ID NO:286, SAKTAVSQRVWLPSHRGGEP; SEQ ID NO:287,KSREHVNNSACPSKRITAAL; SEQ ID NO:288, EGFR; SEQ ID NO:289, RVS, AGS,AGLGVR; SEQ ID NO:290, GGR, GGL, GSV, GVS, GTRQGHTMRLGVSDG; SEQ IDNO:291, IAGLATPGWSHWLAL; SEQ ID NO:292, SMSIARL; SEQ ID NO:293, HTFEPGV;SEQ ID NO:294, NTSLKRISNKRIRRK; SEQ ID NO:295, LRIKRKRRKKTRK; SEQ IDNO:296, GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH and GTV).

As yet a further embodiment, the targeting sequence may be a peptidethat can be used for chemical coupling (e.g., can comprise arginineand/or lysine residues that can be chemically coupled through their Rgroups) to another molecule that targets entry into a cell.

As another embodiment, the AAV capsid protein or virus capsid of theinvention can comprise a mutation as described in WO 2006/066066. Forexample, the capsid protein can comprise a selective amino acidsubstitution at amino acid position 263, 705, 708 and/or 716 of thenative AAV2 capsid protein or a corresponding change(s) in a capsidprotein from another AAV serotype.

Additionally, or alternatively, in representative embodiments, thecapsid protein, virus capsid or vector comprises a selective amino acidinsertion directly following amino acid position 264 of the AAV2 capsidprotein or a corresponding change in the capsid protein from other AAV.By “directly following amino acid position X” it is intended that theinsertion immediately follows the indicated amino acid position (forexample, “following amino acid position 264” indicates a point insertionat position 265 or a larger insertion, e.g., from positions 265 to 268,etc.).

Furthermore, in representative embodiments, the capsid protein, viruscapsid or vector of this invention can comprise amino acid modificationssuch as described in PCT Publication No. WO 2010/093784 (e.g., 2i8)and/or in PCT Publication No. WO 2014/144229 (e.g., dual glycan).

In some embodiments of this invention, the capsid protein, virus capsidor vector of this invention can have equivalent or enhanced transductionefficiency relative to the transduction efficiency of the AAV serotypefrom which the capsid protein, virus capsid or vector of this inventionoriginated. In some embodiments of this invention, the capsid protein,virus capsid or vector of this invention can have reduced transductionefficiency relative to the transduction efficiency of the AAV serotypefrom which the capsid protein, virus capsid or vector of this inventionoriginated. In some embodiments of this invention, the capsid protein,virus capsid or vector of this invention can have equivalent or enhancedtropism relative to the tropism of the AAV serotype from which thecapsid protein, virus capsid or vector of this invention originated. Insome embodiments of this invention, the capsid protein, virus capsid orvector of this invention can have an altered or different tropismrelative to the tropism of the AAV serotype from which the capsidprotein, virus capsid or vector of this invention originated.

In some embodiments of this invention, the capsid protein, virus capsidor vector of this invention can have or be engineered to have tropismfor brain tissue.

The foregoing embodiments of the invention can be used to deliver aheterologous nucleic acid to a cell or subject as described herein. Forexample, the modified vector can be used to treat a lysosomal storagedisorder such as a mucopolysaccharidosis disorder (e.g., Sly syndrome[β-glucuronidase], Hurler Syndrome [α-L-iduronidase], Scheie Syndrome[α-L-iduronidase], Hurler-Scheie Syndrome [α-L-iduronidase], Hunter'sSyndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparansulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:α-glucosaminideacetyltransferase], D [N-acetylglucosamine 6-sulfatase], MorquioSyndrome A [galactose-6-sulfate sulfatase], B [β-galactosidase],Maroteaux-Lamy Syndrome [N-acetylgalactosamine-4-sulfatase], etc.),Fabry disease (α-galactosidase), Gaucher's disease (glucocerebrosidase),or a glycogen storage disorder (e.g., Pompe disease; lysosomal acidα-glucosidase) as described herein.

Those skilled in the art will appreciate that for some AAV capsidproteins the corresponding modification will be an insertion and/or asubstitution, depending on whether the corresponding amino acidpositions are partially or completely present in the virus or,alternatively, are completely absent. Likewise, when modifying AAV otherthan AAV2, the specific amino acid position(s) may be different than theposition in AAV2 (see, e.g., Table 4). As discussed elsewhere herein,the corresponding amino acid position(s) will be readily apparent tothose skilled in the art using well-known techniques.

Nonlimiting examples of corresponding positions in a number of other AAVare shown in Table 4 (Position 2). In particular embodiments, the aminoacid insertion or substitution is a threonine, aspartic acid, glutamicacid or phenylalanine (excepting AAV that have a threonine, glutamicacid or phenylalanine, respectively, at this position).

In other representative embodiments, the modified capsid proteins orvirus capsids of the invention further comprise one or more mutations asdescribed in WO 2007/089632 (e.g., an E→K mutation at amino acidposition 531 of the AAV2 capsid protein or the corresponding position ofthe capsid protein from another AAV).

In further embodiments, the modified capsid protein or capsid cancomprise a mutation as described in WO 2009/108274.

As another, possibility, the AAV capsid protein can comprise a mutationas described by Zhong et al. (Virology 381: 194-202 (2008); Proc. Nat.Acad Sci. 105: 7827-32 (2008)). For example, the AAV capsid protein cancomprise a Y→F mutation at amino acid position 730.

The modifications described above can be incorporated into the capsidproteins or capsids of the invention in combination with each otherand/or with any other modification now known or later discovered.

TABLE 4 Serotype Position 1 Position 2 AAV1 A263X T265X AAV2 Q263X —265XAAV3A Q263X —265X AAV3B Q263X —265X AAV4 S257X —259X AAV5 G253X V255XAAV6 A263X T265X AAV7 E264X A266X AAV8 G264X S266X AAV9 S263X S265XWhere, (X) → mutation to any amino acid; (—) → insertion of any aminoacid Note: Position 2 inserts are indicated by the site of insertion

The invention also encompasses virus vectors comprising the modifiedcapsid proteins and capsids of the invention. In particular embodiments,the virus vector is a parvovirus vector (e.g., comprising a parvoviruscapsid and/or vector genome), for example, an AAV vector (e.g.,comprising an AAV capsid and/or vector genome). In representativeembodiments, the virus vector comprises a modified AAV capsid comprisinga modified capsid subunit of the invention and a vector genome.

For example, in representative embodiments, the virus vector comprises:(a) a modified virus capsid (e.g., a modified AAV capsid) comprising amodified capsid protein of the invention; and (b) a nucleic acidcomprising a terminal repeat sequence (e.g., an AAV TR), wherein thenucleic acid comprising the terminal repeat sequence is encapsidated bythe modified virus capsid. The nucleic acid can optionally comprise twoterminal repeats (e.g., two AAV TRs).

In representative embodiments, the virus vector is a recombinant virusvector comprising a heterologous nucleic acid encoding a polypeptide orfunctional RNA of interest. Recombinant virus vectors are described inmore detail below.

In particular embodiments, the virus vectors of the invention (i) havereduced transduction of liver as compared with the level of transductionby a virus vector without the modified capsid protein; (ii) exhibitenhanced systemic transduction by the virus vector in an animal subjectas compared with the level observed by a virus vector without themodified capsid protein; (iii) demonstrate enhanced movement acrossendothelial cells as compared with the level of movement by a virusvector without the modified capsid protein, and/or (iv) exhibit aselective enhancement in transduction of muscle tissue (e.g., skeletalmuscle, cardiac muscle and/or diaphragm muscle), and/or (v) reducedtransduction of brain tissues (e.g., neurons) as compared with the levelof transduction by a virus vector without the modified capsid protein.In particular embodiments, the virus vector has systemic transductiontoward muscle, e.g., transduces multiple skeletal muscle groupsthroughout the body and optionally transduces cardiac muscle and/ordiaphragm muscle.

It will be understood by those skilled in the art that the modifiedcapsid proteins, virus capsids and virus vectors of the inventionexclude those capsid proteins, capsids and virus vectors that have theindicated amino acids at the specified positions in their native state(i.e., are not mutants).

Methods of Producing Virus Vectors.

The present invention further provides methods of producing theinventive virus vectors. Thus, in one embodiment, the present inventionprovides a method of producing an AAV vector that evades neutralizingantibodies, comprising: a) identifying contact amino acid residues thatform a three dimensional antigenic footprint on an AAV capsid protein;b) generating a library of AAV capsid proteins comprising amino acidsubstitutions of the contact amino acid residues identified in (a); c)producing AAV particles comprising capsid proteins from the library ofAAV capsid proteins of (b); d) contacting the AAV particles of (c) withcells under conditions whereby infection and replication can occur; e)selecting AAV particles that can complete at least one infectious cycleand replicate to titers similar to control AAV particles; f) contactingthe AAV particles selected in (e) with neutralizing antibodies and cellsunder conditions whereby infection and replication can occur; and g)selecting AAV particles that are not neutralized by the neutralizingantibodies of (f) Nonlimiting examples of methods for identifyingcontact amino acid residues include peptide epitope mapping and/orcryo-electron microscopy.

Resolution and identification of the antibody contact residues withinthe three dimensional antigenic footprint allows for their subsequentmodification through random, rational and/or degenerate mutagenesis togenerate antibody-evading AAV capsids that can be identified throughfurther selection and/or screening.

Thus, in a further embodiment, the present invention provides a methodof producing an AAV vector that evades neutralizing antibodies,comprising: a) identifying contact amino acid residues that form a threedimensional antigenic footprint on an AAV capsid protein; b) generatingAAV capsid proteins comprising amino acid substitutions of the contactamino acid residues identified in (a) by random, rational and/ordegenerate mutagenesis; c) producing AAV particles comprising capsidproteins from the AAV capsid proteins of (b); d) contacting the AAVparticles of (c) with cells under conditions whereby infection andreplication can occur, e) selecting AAV particles that can complete atleast one infectious cycle and replicate to titers similar to controlAAV particles; f) contacting the AAV particles selected in (e) withneutralizing antibodies and cells under conditions whereby infection andreplication can occur, and g) selecting AAV particles that are notneutralized by the neutralizing antibodies of (f)

Nonlimiting examples of methods for identifying contact amino acidresidues include peptide epitope mapping and/or cryo-electronmicroscopy. Methods of generating AAV capsid proteins comprising aminoacid substitutions of contact amino acid residues by random, rationaland/or degenerate mutagenesis are known in the art.

This comprehensive approach presents a platform technology that can beapplied to modifying any AAV capsid. Application of this platformtechnology yields AAV antigenic variants derived from the original AAVcapsid template without loss of transduction efficiency. As oneadvantage and benefit, application of this technology will expand thecohort of patients eligible for gene therapy with AAV vectors.

In one embodiment, the present invention provides a method of producinga virus vector, the method comprising providing to a cell: (a) a nucleicacid template comprising at least one TR sequence (e.g., AAV TRsequence), and (b) AAV sequences sufficient for replication of thenucleic acid template and encapsidation into AAV capsids (e.g., AAV repsequences and AAV cap sequences encoding the AAV capsids of theinvention). Optionally, the nucleic acid template further comprises atleast one heterologous nucleic acid sequence. In particular embodiments,the nucleic acid template comprises two AAV ITR sequences, which arelocated 5′ and 3′ to the heterologous nucleic acid sequence (ifpresent), although they need not be directly contiguous thereto.

The nucleic acid template and AAV rep and cap sequences are providedunder conditions such that virus vector comprising the nucleic acidtemplate packaged within the AAV capsid is produced in the cell. Themethod can further comprise the step of collecting the virus vector fromthe cell. The virus vector can be collected from the medium and/or bylysing the cells.

The cell can be a cell that is permissive for AAV viral replication. Anysuitable cell known in the art may be employed. In particularembodiments, the cell is a mammalian cell. As another option, the cellcan be a trans-complementing packaging cell line that provides functionsdeleted from a replication-defective helper virus, e.g., 293 cells orother E1a trans-complementing cells.

The AAV replication and capsid sequences may be provided by any methodknown in the art. Current protocols typically express the AAV rep/capgenes on a single plasmid. The AAV replication and packaging sequencesneed not be provided together, although it may be convenient to do so.The AAV rep and/or cap sequences may be provided by any viral ornon-viral vector. For example, the rep/cap sequences may be provided bya hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1aor E3 regions of a deleted adenovirus vector). EBV vectors may also beemployed to express the AAV cap and rep genes. One advantage of thismethod is that EBV vectors are episomal, yet will maintain a high copynumber throughout successive cell divisions (i.e., are stably integratedinto the cell as extra-chromosomal elements, designated as an “EBV basednuclear episome,” see Margolski, (1992) Curr. Top. Microbiol. Immun.158:67).

As a further alternative, the rep/cap sequences may be stablyincorporated into a cell.

Typically the AAV rep/cap sequences will not be flanked by the TRs, toprevent rescue and/or packaging of these sequences.

The nucleic acid template can be provided to the cell using any methodknown in the art. For example, the template can be supplied by anon-viral (e.g., plasmid) or viral vector. In particular embodiments,the nucleic acid template is supplied by a herpesvirus or adenovirusvector (e.g., inserted into the E1a or E3 regions of a deletedadenovirus). As another illustration, Palombo et al., (1998) J. Virology72:5025, describes a baculovirus vector carrying a reporter gene flankedby the AAV TRs. EBV vectors may also be employed to deliver thetemplate, as described above with respect to the rep/cap genes.

In another representative embodiment, the nucleic acid template isprovided by a replicating rAAV virus. In still other embodiments, an AAVprovirus comprising the nucleic acid template is stably integrated intothe chromosome of the cell.

To enhance virus titers, helper virus functions (e.g., adenovirus orherpesvirus) that promote a productive AAV infection can be provided tothe cell. Helper virus sequences necessary for AAV replication are knownin the art. Typically, these sequences will be provided by a helperadenovirus or herpesvirus vector. Alternatively, the adenovirus orherpesvirus sequences can be provided by another non-viral or viralvector, e.g., as a non-infectious adenovirus miniplasmid that carriesall of the helper genes that promote efficient AAV production asdescribed by Ferrari et al., (1997) Nature Med 3:1295, and U.S. Pat.Nos. 6,040,183 and 6,093,570.

Further, the helper virus functions may be provided by a packaging cellwith the helper sequences embedded in the chromosome or maintained as astable extrachromosomal element. Generally, the helper virus sequencescannot be packaged into AAV virions, e.g., are not flanked by TRs.

Those skilled in the art will appreciate that it may be advantageous toprovide the AAV replication and capsid sequences and the helper virussequences (e.g., adenovirus sequences) on a single helper construct.This helper construct may be a non-viral or viral construct. As onenonlimiting illustration, the helper construct can be a hybridadenovirus or hybrid herpesvirus comprising the AAV rep/cap genes.

In one particular embodiment, the AAV rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. This vector further can further comprise the nucleic acidtemplate. The AAV rep/cap sequences and/or the rAAV template can beinserted into a deleted region (e.g., the E1a or E3 regions) of theadenovirus.

In a further embodiment, the AAV rep/cap sequences and the adenovirushelper sequences are supplied by a single adenovirus helper vector.According to this embodiment, the rAAV template can be provided as aplasmid template.

In another illustrative embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helpervector, and the rAAV template is integrated into the cell as a provirus.Alternatively, the rAAV template is provided by an EBV vector that ismaintained within the cell as an extrachromosomal element (e.g., as anEBV based nuclear episome).

In a further exemplary embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helper.The rAAV template can be provided as a separate replicating viralvector. For example, the rAAV template can be provided by a rAAVparticle or a second recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vectortypically comprises the adenovirus 5′ and 3′ cis sequences sufficientfor adenovirus replication and packaging (i.e., the adenovirus terminalrepeats and PAC sequence). The AAV rep/cap sequences and, if present,the rAAV template are embedded in the adenovirus backbone and areflanked by the 5′ and 3′ cis sequences, so that these sequences may bepackaged into adenovirus capsids. As described above, the adenovirushelper sequences and the AAV rep/cap sequences are generally not flankedby TRs so that these sequences are not packaged into the AAV virions.

Zhang et al., ((2001) Gene Ther. 18:704-12) describe a chimeric helpercomprising both adenovirus and the AAV rep and cap genes.

Herpesvirus may also be used as a helper virus in AAV packaging methods.Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageouslyfacilitate scalable AAV vector production schemes. A hybrid herpessimplex virus type I (HSV-1) vector expressing the AAV-2 rep and capgenes has been described (Conway et al., (1999) Gene Therapy 6:986 andWO 00/17377.

As a further alternative, the virus vectors of the invention can beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and rAAV template as described, for example, by Urabe etal., (2002) Human Gene Therapy 13:1935-43.

AAV vector stocks free of contaminating helper virus may be obtained byany method known in the art. For example, AAV and helper virus may bereadily differentiated based on size. AAV may also be separated awayfrom helper virus based on affinity for a heparin substrate (Zolotukhinet al. (1999) Gene Therapy 6:973). Deleted replication-defective helperviruses can be used so that any contaminating helper virus is notreplication competent. As a further alternative, an adenovirus helperlacking late gene expression may be employed, as only adenovirus earlygene expression is required to mediate packaging of AAV virus.Adenovirus mutants defective for late gene expression are known in theart (e.g., ts100K and ts149 adenovirus mutants).

Recombinant Virus Vectors.

The virus vectors of the present invention are useful for the deliveryof nucleic acids to cells in vitro, ex vivo, and in vivo. In particular,the virus vectors can be advantageously employed to deliver or transfernucleic acids to animal, including mammalian, cells.

Any heterologous nucleic acid sequence(s) of interest may be deliveredin the virus vectors of the present invention. Nucleic acids of interestinclude nucleic acids encoding polypeptides, including therapeutic(e.g., for medical or veterinary uses) or immunogenic (e.g., forvaccines) polypeptides.

Therapeutic polypeptides include, but are not limited to, cysticfibrosis transmembrane regulator protein (CFTR), dystrophin (includingmini- and micro-dystrophins, see, e.g., Vincent et al., (1993) NatureGenetics 5:130; U.S. Patent Publication No. 2003/017131; Internationalpublication WO/2008/088895, Wang et al., Proc. Natl. Acad Sci. USA97:13714-13719 (2000); and Gregorevic et al., Mol. Ther. 16:657-64(2008)), myostatin propeptide, follistatin, activin type II solublereceptor, IGF-1, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, utrophin (Tinsley et al., (1996) Nature384:349), mini-utrophin, clotting factors (e.g., Factor VIII, Factor IX,Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase,tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor,lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin,spectrin, α₁-antitrypsin, adenosine deaminase, hypoxanthine guaninephosphoribosyl transferase, β-glucocerebrosidase, sphingomyelinase,lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase, RP65protein, cytokines (e.g., α-interferon, β-interferon, interferon-γ,interleukin-2, interleukin-4, granulocyte-macrophage colony stimulatingfactor, lymphotoxin, and the like), peptide growth factors, neurotrophicfactors and hormones (e.g., somatotropin, insulin, insulin-like growthfactors 1 and 2, platelet derived growth factor, epidermal growthfactor, fibroblast growth factor, nerve growth factor, neurotrophicfactor-3 and -4, brain-derived neurotrophic factor, bone morphogenicproteins [including RANKL and VEGF], glial derived growth factor,transforming growth factor-α and -β, and the like), lysosomal acidα-glucosidase, α-galactosidase A, receptors (e.g., the tumor necrosisgrowth factor α soluble receptor), S100A1, parvalbumin, adenylyl cyclasetype 6, a molecule that modulates calcium handling (e.g., SERCA_(2A),Inhibitor 1 of PP1 and fragments thereof [e.g., WO 2006/029319 and WO2007/100465]), a molecule that effects G-protein coupled receptor kinasetype 2 knockdown such as a truncated constitutively active bARKct,anti-inflammatory factors such as IRAP, anti-myostatin proteins,aspartoacylase, monoclonal antibodies (including single chain monoclonalantibodies; an exemplary Mab is the Herceptin® Mab), neuropeptides andfragments thereof (e.g., galanin, Neuropeptide Y (see, U.S. Pat. No.7,071,172), angiogenesis inhibitors such as Vasohibins and other VEGFinhibitors (e.g., Vasohibin 2 [see, WO JP2006/073052]). Otherillustrative heterologous nucleic acid sequences encode suicide geneproducts (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin,and tumor necrosis factor), proteins conferring resistance to a drugused in cancer therapy, tumor suppressor gene products (e.g., p53, Rb,Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has atherapeutic effect in a subject in need thereof. AAV vectors can also beused to deliver monoclonal antibodies and antibody fragments, forexample, an antibody or antibody fragment directed against myostatin(see, e.g., Fang et al., Nature Biotechnology 23:584-590 (2005)).

Heterologous nucleic acid sequences encoding polypeptides include thoseencoding reporter polypeptides (e.g., an enzyme). Reporter polypeptidesare known in the art and include, but are not limited to, GreenFluorescent Protein, β-galactosidase, alkaline phosphatase, luciferase,and chloramphenicol acetyltransferase gene.

Optionally, the heterologous nucleic acid encodes a secreted polypeptide(e.g., a polypeptide that is a secreted polypeptide in its native stateor that has been engineered to be secreted, for example, by operableassociation with a secretory signal sequence as is known in the art).

Alternatively, in particular embodiments of this invention, theheterologous nucleic acid may encode an antisense nucleic acid, aribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs thateffect spliceosome-mediated trans-splicing (see, Puttaraju et al.,(1999) Nature Biotech. 17:246; U.S. Pat. Nos. 6,013,487; 6,083,702),interfering RNAs (RNAi) including siRNA, shRNA or miRNA that mediategene silencing (see, Sharp et al., (2000) Science 287:2431), and othernon-translated RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc.Nat. Acad Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), andthe like. Exemplary untranslated RNAs include RNAi against a multipledrug resistance (MDR) gene product (e.g., to treat and/or prevent tumorsand/or for administration to the heart to prevent damage bychemotherapy), RNAi against myostatin (e.g., for Duchenne musculardystrophy), RNAi against VEGF (e.g., to treat and/or prevent tumors),RNAi against phospholamban (e.g., to treat cardiovascular disease, see,e.g., Andino et al., J. Gene Med 10:132-142 (2008) and Li et al., ActaPharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E (e.g., to treatcardiovascular disease, see, e.g., Hoshijima et al. Nat. Med 8:864-871(2002)), RNAi to adenosine kinase (e.g., for epilepsy), and RNAidirected against pathogenic organisms and viruses (e.g., hepatitis Band/or C virus, human immunodeficiency virus, CMV, herpes simplex virus,human papilloma virus, etc.).

Further, a nucleic acid sequence that directs alternative splicing canbe delivered. To illustrate, an antisense sequence (or other inhibitorysequence) complementary to the 5′ and/or 3′ splice site of dystrophinexon 51 can be delivered in conjunction with a U1 or U7 small nuclear(sn) RNA promoter to induce skipping of this exon. For example, a DNAsequence comprising a U1 or U7 snRNA promoter located 5′ to theantisense/inhibitory sequence(s) can be packaged and delivered in amodified capsid of the invention.

The virus vector may also comprise a heterologous nucleic acid thatshares homology with and recombines with a locus on a host chromosome.This approach can be utilized, for example, to correct a genetic defectin the host cell.

The present invention also provides virus vectors that express animmunogenic polypeptide, e.g., for vaccination. The nucleic acid mayencode any immunogen of interest known in the art including, but notlimited to, immunogens from human immunodeficiency virus (HIV), simianimmunodeficiency virus (SW), influenza virus, HIV or SIV gag proteins,tumor antigens, cancer antigens, bacterial antigens, viral antigens, andthe like.

The use of parvoviruses as vaccine vectors is known in the art (see,e.g., Miyamura et al., (1994) Proc. Nat. Acad Sci USA 91:8507; U.S. Pat.No. 5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara etal., U.S. Pat. Nos. 5,882,652, 5,863,541 to Samulski et al.). Theantigen may be presented in the parvovirus capsid. Alternatively, theantigen may be expressed from a heterologous nucleic acid introducedinto a recombinant vector genome. Any immunogen of interest as describedherein and/or as is known in the art can be provided by the virus vectorof the present invention.

An immunogenic polypeptide can be any polypeptide suitable for elicitingan immune response and/or protecting the subject against an infectionand/or disease, including, but not limited to, microbial, bacterial,protozoal, parasitic, fungal and/or viral infections and diseases. Forexample, the immunogenic polypeptide can be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoprotein,or an equine influenza virus immunogen) or a lentivirus immunogen (e.g.,an equine infectious anemia virus immunogen, a Simian ImmunodeficiencyVirus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV)immunogen, such as the HIV or SIV envelope GP160 protein, the HIV or SIVmatrix/capsid proteins, and the HIV or SIV gag, pol and env genesproducts). The immunogenic polypeptide can also be an arenavirusimmunogen (e.g., Lassa fever virus immunogen, such as the Lassa fevervirus nucleocapsid protein and the Lassa fever envelope glycoprotein), apoxvirus immunogen (e.g., a vaccinia virus immunogen, such as thevaccinia L1 or L8 gene products), a flavivirus immunogen (e.g., a yellowfever virus immunogen or a Japanese encephalitis virus immunogen), afilovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virusimmunogen, such as NP and GP gene products), a bunyavirus immunogen(e.g., RVFV, CCHF, and/or SFS virus immunogens), or a coronavirusimmunogen (e.g., an infectious human coronavirus immunogen, such as thehuman coronavirus envelope glycoprotein, or a porcine transmissiblegastroenteritis virus immunogen, or an avian infectious bronchitis virusimmunogen). The immunogenic polypeptide can further be a polioimmunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens) a mumpsimmunogen, a measles immunogen, a rubella immunogen, a diphtheria toxinor other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g.,hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and/or any othervaccine immunogen now known in the art or later identified as animmunogen.

Alternatively, the immunogenic polypeptide can be any tumor or cancercell antigen. Optionally, the tumor or cancer antigen is expressed onthe surface of the cancer cell. Exemplary cancer and tumor cell antigensare described in S. A. Rosenberg (Immunity 10:281 (1991)). Otherillustrative cancer and tumor antigens include, but are not limited to:BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2,BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, Caspase-8,KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami etal., (1994) Proc. Natl. Acad Sci. USA 91:3515; Kawakami et al., (1994)J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124),MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15,tyrosinase (Brichard et al, (1993) J. Exp. Med. 178:489); HER-2/neu geneproduct (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialylTn antigen), c-erbB-2 proteins, PSA, L-CanAg, estrogen receptor, milkfat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev.Biochem. 62:623); mucin antigens (International Patent Publication No.WO 90/05142); telomerases; nuclear matrix proteins; prostatic acidphosphatase; papilloma virus antigens; and/or antigens now known orlater discovered to be associated with the following cancers: melanoma,adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma,Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer,leukemia, uterine cancer, breast cancer, prostate cancer, ovariancancer, cervical cancer, bladder cancer, kidney cancer, pancreaticcancer, brain cancer and any other cancer or malignant condition nowknown or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med.47:481-91).

As a further alternative, the heterologous nucleic acid can encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed gene product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest can be operably associated with appropriatecontrol sequences. For example, the heterologous nucleic acid can beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, and/or enhancers, and the like.

Further, regulated expression of the heterologous nucleic acid(s) ofinterest can be achieved at the post-transcriptional level, e.g., byregulating selective splicing of different introns by the presence orabsence of an oligonucleotide, small molecule and/or other compound thatselectively blocks splicing activity at specific sites (e.g., asdescribed in WO 2006/119137).

Those skilled in the art will appreciate that a variety ofpromoter/enhancer elements can be used depending on the level andtissue-specific expression desired. The promoter/enhancer can beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

In particular embodiments, the promoter/enhancer elements can be nativeto the target cell or subject to be treated. In representativeembodiments, the promoters/enhancer element can be native to theheterologous nucleic acid sequence. The promoter/enhancer element isgenerally chosen so that it functions in the target cell(s) of interest.Further, in particular embodiments the promoter/enhancer element is amammalian promoter/enhancer element. The promoter/enhancer element maybe constitutive or inducible.

Inducible expression control elements are typically advantageous inthose applications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific or-preferred promoter/enhancer elements, and include muscle specific orpreferred (including cardiac, skeletal and/or smooth muscle specific orpreferred), neural tissue specific or preferred (includingbrain-specific or preferred), eye specific or preferred (includingretina-specific and cornea-specific), liver specific or preferred, bonemarrow specific or preferred, pancreatic specific or preferred, spleenspecific or preferred, and lung specific or preferred promoter/enhancerelements. Other inducible promoter/enhancer elements includehormone-inducible and metal-inducible elements. Exemplary induciblepromoters/enhancer elements include, but are not limited to, a Teton/off element, a RU486-inducible promoter, an ecdysone-induciblepromoter, a rapamycin-inducible promoter, and a metallothioneinpromoter.

In embodiments wherein the heterologous nucleic acid sequence(s) istranscribed and then translated in the target cells, specific initiationsignals are generally included for efficient translation of insertedprotein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The virus vectors according to the present invention provide a means fordelivering heterologous nucleic acids into a broad range of cells,including dividing and non-dividing cells. The virus vectors can beemployed to deliver a nucleic acid of interest to a cell in vitro, e.g.,to produce a polypeptide in vitro or for ex vivo gene therapy. The virusvectors are additionally useful in a method of delivering a nucleic acidto a subject in need thereof, e.g., to express an immunogenic ortherapeutic polypeptide or a functional RNA. In this manner, thepolypeptide or functional RNA can be produced in vivo in the subject.The subject can be in need of the polypeptide because the subject has adeficiency of the polypeptide. Further, the method can be practicedbecause the production of the polypeptide or functional RNA in thesubject may impart some beneficial effect.

The virus vectors can also be used to produce a polypeptide of interestor functional RNA in cultured cells or in a subject (e.g., using thesubject as a bioreactor to produce the polypeptide or to observe theeffects of the functional RNA on the subject, for example, in connectionwith screening methods).

In general, the virus vectors of the present invention can be employedto deliver a heterologous nucleic acid encoding a polypeptide orfunctional RNA to treat and/or prevent any disease state for which it isbeneficial to deliver a therapeutic polypeptide or functional RNA.Illustrative disease states include, but are not limited to: cysticfibrosis (cystic fibrosis transmembrane regulator protein) and otherdiseases of the lung, hemophilia A (Factor VIII), hemophilia B (FactorIX), thalassemia (ß-globin), anemia (erythropoietin) and other blooddisorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis(ß-interferon), Parkinson's disease (glial-cell line derivedneurotrophic factor [GDNF]), Huntington's disease (RNAi to removerepeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophicfactors), and other neurological disorders, cancer (endostatin,angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAiincluding RNAi against VEGF or the multiple drug resistance geneproduct, mir-26a [e.g., for hepatocellular carcinoma]), diabetesmellitus (insulin), muscular dystrophies including Duchenne (dystrophin,mini-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., α,β, γ], RNAi against myostatin, myostatin propeptide, follistatin,activin type II soluble receptor, anti-inflammatory polypeptides such asthe Ikappa B dominant mutant, sarcospan, utrophin, mini-utrophin,antisense or RNAi against splice junctions in the dystrophin gene toinduce exon skipping [see, e.g., WO/2003/095647], antisense against U7snRNAs to induce exon skipping [see, e.g., WO/2006/021724], andantibodies or antibody fragments against myostatin or myostatinpropeptide) and Becker, Gaucher disease (glucocerebrosidase), Hurler'sdisease (α-L-iduronidase), adenosine deaminase deficiency (adenosinedeaminase), glycogen storage diseases (e.g., Fabry disease[α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) andother metabolic disorders, congenital emphysema (α1-antitrypsin),Lesch-Nyhan Syndrome (hypoxanthine guanine phosphoribosyl transferase),Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomalhexosaminidase A), Maple Syrup Urine Disease (branched-chain keto aciddehydrogenase), retinal degenerative diseases (and other diseases of theeye and retina; e.g., PDGF for macular degeneration and/or vasohibin orother inhibitors of VEGF or other angiogenesis inhibitors totreat/prevent retinal disorders, e.g., in Type I diabetes), diseases ofsolid organs such as brain (including Parkinson's Disease [GDNF],astrocytomas [endostatin, angiostatin and/or RNAi against VEGF],glioblastomas [endostatin, angiostatin and/or RNAi against VEGF]),liver, kidney, heart including congestive heart failure or peripheralartery disease (PAD) (e.g., by delivering protein phosphatase inhibitorI (I-1) and fragments thereof (e.g., I1C), serca2a, zinc finger proteinsthat regulate the phospholamban gene, Barkct, β2-adrenergic receptor,β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3kinase), S100A1, parvalbumin, adenylyl cyclase type 6, a molecule thateffects G-protein coupled receptor kinase type 2 knockdown such as atruncated constitutively active bARKct; calsarcin, RNAi againstphospholamban; phospholamban inhibitory or dominant-negative moleculessuch as phospholamban S16E, etc.), arthritis (insulin-like growthfactors), joint disorders (insulin-like growth factor 1 and/or 2),intimal hyperplasia (e.g., by delivering enos, inos), improve survivalof heart transplants (superoxide dismutase), AIDS (soluble CD4), musclewasting (insulin-like growth factor I), kidney deficiency(erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatoryfactors such as IRAP and TNFα soluble receptor), hepatitis(α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia(omithine transcarbamylase), Krabbe's disease (galactocerebrosidase),Batten's disease, spinal cerebral ataxias including SCA1, SCA2 and SCA3,phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, andthe like. The invention can further be used following organtransplantation to increase the success of the transplant and/or toreduce the negative side effects of organ transplantation or adjuncttherapies (e.g., by administering immunosuppressant agents or inhibitorynucleic acids to block cytokine production). As another example, bonemorphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF) canbe administered with a bone allograft, for example, following a break orsurgical removal in a cancer patient.

The invention can also be used to produce induced pluripotent stem cells(iPS). For example, a virus vector of the invention can be used todeliver stem cell associated nucleic acid(s) into a non-pluripotentcell, such as adult fibroblasts, skin cells, liver cells, renal cells,adipose cells, cardiac cells, neural cells, epithelial cells,endothelial cells, and the like. Nucleic acids encoding factorsassociated with stem cells are known in the art. Nonlimiting examples ofsuch factors associated with stem cells and pluripotency includeOct-3/4, the SOX family (e.g., SOX1, SOX2, SOX3 and/or SOX15), the Klffamily (e.g., Klf1, Klf2, Klf4 and/or Klf5), the Myc family (e.g.,C-myc, L-myc and/or N-myc), NANOG and/or LIN28.

The invention can also be practiced to treat and/or prevent a metabolicdisorder such as diabetes (e.g., insulin), hemophilia (e.g., Factor IXor Factor VIII), a lysosomal storage disorder such as amucopolysaccharidosis disorder (e.g., Sly syndrome [β-glucuronidase],Hurler Syndrome [α-L-iduronidase], Scheie Syndrome [α-L-iduronidase],Hurler-Scheie Syndrome [α-L-iduronidase], Hunter's Syndrome [iduronatesulfatase], Sanfilippo Syndrome A [heparan sulfamidase], B[N-acetylglucosaminidase], C [acetyl-CoA:α-glucosaminideacetyltransferase], D [N-acetylglucosamine 6-sulfatase], MorquioSyndrome A [galactose-6-sulfate sulfatase], B [β-galactosidase],Maroteaux-Lamy Syndrome [N-acetylgalactosamine-4-sulfatase], etc.),Fabry disease (α-galactosidase), Gaucher's disease (glucocerebrosidase),or a glycogen storage disorder (e.g., Pompe disease; lysosomal acidα-glucosidase).

Gene transfer has substantial potential use for understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer can be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using antisensemutations. For unbalanced disease states, gene transfer can be used tocreate a disease state in a model system, which can then be used inefforts to counteract the disease state. Thus, virus vectors accordingto the present invention permit the treatment and/or prevention ofgenetic diseases.

The virus vectors according to the present invention may also beemployed to provide a functional RNA to a cell in vitro or in vivo.Expression of the functional RNA in the cell, for example, can diminishexpression of a particular target protein by the cell. Accordingly,functional RNA can be administered to decrease expression of aparticular protein in a subject in need thereof. Functional RNA can alsobe administered to cells in vitro to regulate gene expression and/orcell physiology, e.g., to optimize cell or tissue culture systems or inscreening methods.

In addition, virus vectors according to the instant invention find usein diagnostic and screening methods, whereby a nucleic acid of interestis transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model.

The virus vectors of the present invention can also be used for variousnon-therapeutic purposes, including but not limited to use in protocolsto assess gene targeting, clearance, transcription, translation, etc.,as would be apparent to one skilled in the art. The virus vectors canalso be used for the purpose of evaluating safety (spread, toxicity,immunogenicity, etc.). Such data, for example, are considered by theUnited States Food and Drug Administration as part of the regulatoryapproval process prior to evaluation of clinical efficacy.

As a further aspect, the virus vectors of the present invention may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a heterologous nucleic acidsequence encoding an immunogenic polypeptide can be administered to asubject, and an active immune response is mounted by the subject againstthe immunogenic polypeptide. Immunogenic polypeptides are as describedhereinabove. In some embodiments, a protective immune response iselicited.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The virus vectorcomprising the heterologous nucleic acid is introduced into the cell,and the cell is administered to the subject, where the heterologousnucleic acid encoding the immunogen can be expressed and induce animmune response in the subject against the immunogen. In particularembodiments, the cell is an antigen-presenting cell (e.g., a dendriticcell).

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure to animmunogen by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment and/or prevention of disease, in particular cancer or tumors(e.g., by preventing cancer or tumor formation, by causing regression ofa cancer or tumor and/or by preventing metastasis and/or by preventinggrowth of metastatic nodules). The protective effects may be complete orpartial, as long as the benefits of the treatment outweigh anydisadvantages thereof.

In particular embodiments, the virus vector or cell comprising theheterologous nucleic acid can be administered in an immunogenicallyeffective amount, as described below.

The virus vectors of the present invention can also be administered forcancer immunotherapy by administration of a virus vector expressing oneor more cancer cell antigens (or an immunologically similar molecule) orany other immunogen that produces an immune response against a cancercell. To illustrate, an immune response can be produced against a cancercell antigen in a subject by administering a virus vector comprising aheterologous nucleic acid encoding the cancer cell antigen, for exampleto treat a patient with cancer and/or to prevent cancer from developingin the subject. The virus vector may be administered to a subject invive or by using ex vive methods, as described herein. Alternatively,the cancer antigen can be expressed as part of the virus capsid or beotherwise associated with the virus capsid (e.g., as described above).

As another alternative, any other therapeutic nucleic acid (e.g., RNAi)or polypeptide (e.g., cytokine) known in the art can be administered totreat and/or prevent cancer.

As used herein, the term “cancer” encompasses tumor-forming cancers.Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cellantigen” encompasses tumor antigens.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to melanoma, adenocarcinoma, thymoma,lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma,lung cancer, liver cancer, colon cancer, leukemia, uterine cancer,breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladdercancer, kidney cancer, pancreatic cancer, brain cancer and any othercancer or malignant condition now known or later identified. Inrepresentative embodiments, the invention provides a method of treatingand/or preventing tumor-forming cancers.

The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multicellular organism.Tumors can be malignant or benign. In representative embodiments, themethods disclosed herein are used to prevent and treat malignant tumors.

By the terms “treating cancer,” “treatment of cancer” and equivalentterms it is intended that the severity of the cancer is reduced or atleast partially eliminated and/or the progression of the disease isslowed and/or controlled and/or the disease is stabilized. In particularembodiments, these terms indicate that metastasis of the cancer isprevented or reduced or at least partially eliminated and/or that growthof metastatic nodules is prevented or reduced or at least partiallyeliminated.

By the terms “prevention of cancer” or “preventing cancer” andequivalent terms it is intended that the methods at least partiallyeliminate or reduce and/or delay the incidence and/or severity of theonset of cancer. Alternatively stated, the onset of cancer in thesubject may be reduced in likelihood or probability and/or delayed.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector expressing a cancer cellantigen according to the instant invention. The modified cell is thenadministered to the subject, whereby an immune response against thecancer cell antigen is elicited. This method can be advantageouslyemployed with immunocompromised subjects that cannot mount a sufficientimmune response in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, immunomodulatory cytokines (preferably, CTLinductive cytokines) may be administered to a subject in conjunctionwith the virus vector.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleic acid encoding a cytokine may be delivered to the subject using asuitable vector, and the cytokine produced in vivo.

Subjects, Pharmaceutical Formulations, and Modes of Administration.

Virus vectors and capsids according to the present invention find use inboth veterinary and medical applications. Suitable subjects include bothavians and mammals. The term “avian” as used herein includes, but is notlimited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots,parakeets, and the like. The term “mammal” as used herein includes, butis not limited to, humans, non-human primates, bovines, ovines,caprines, equines, felines, canines, lagomorphs, etc. Human subjectsinclude neonates, infants, juveniles, adults and geriatric subjects.

In representative embodiments, the subject is “in need of” the methodsof the invention.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector and/or capsidand/or capsid protein and/or virus particle of the invention in apharmaceutically acceptable carrier and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. For other methods of administration, the carrier may be eithersolid or liquid. For inhalation administration, the carrier will berespirable, and optionally can be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring anucleic acid to a cell in vitro. The virus vector may be introduced intothe cells at the appropriate multiplicity of infection according tostandard transduction methods suitable for the particular target cells.Titers of virus vector to administer can vary, depending upon the targetcell type and number, and the particular virus vector, and can bedetermined by those of skill in the art without undue experimentation.In representative embodiments, at least about 10³ infectious units,optionally at least about 10⁵ infectious units are introduced to thecell.

The cell(s) into which the virus vector is introduced can be of anytype, including but not limited to neural cells (including cells of theperipheral and central nervous systems, in particular, brain cells suchas neurons and oligodendricytes), lung cells, cells of the eye(including retinal cells, retinal pigment epithelium, and cornealcells), epithelial cells (e.g., gut and respiratory epithelial cells),muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smoothmuscle cells and/or diaphragm muscle cells), dendritic cells, pancreaticcells (including islet cells), hepatic cells, myocardial cells, bonecells (e.g., bone marrow stem cells), hematopoietic stem cells, spleencells, keratinocytes, fibroblasts, endothelial cells, prostate cells,germ cells, and the like. In representative embodiments, the cell can beany progenitor cell. As a further possibility, the cell can be a stemcell (e.g., neural stem cell, liver stem cell). As still a furtheralternative, the cell can be a cancer or tumor cell. Moreover, the cellcan be from any species of origin, as indicated above.

The virus vector can be introduced into cells in vitro for the purposeof administering the modified cell to a subject. In particularembodiments, the cells have been removed from a subject, the virusvector is introduced therein, and the cells are then administered backinto the subject. Methods of removing cells from subject formanipulation ex vivo, followed by introduction back into the subject areknown in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively,the recombinant virus vector can be introduced into cells from a donorsubject, into cultured cells, or into cells from any other suitablesource, and the cells are administered to a subject in need thereof(i.e., a “recipient” subject).

Suitable cells for ex vivo nucleic acid delivery are as described above.Dosages of the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ cells or at least about 10³to about 10⁶ cells will be administered per dose in a pharmaceuticallyacceptable carrier. In particular embodiments, the cells transduced withthe virus vector are administered to the subject in a treatmenteffective or prevention effective amount in combination with apharmaceutical carrier.

In some embodiments, the virus vector is introduced into a cell and thecell can be administered to a subject to elicit an immunogenic responseagainst the delivered polypeptide (e.g., expressed as a transgene or inthe capsid). Typically, a quantity of cells expressing animmunogenically effective amount of the polypeptide in combination witha pharmaceutically acceptable carrier is administered. An“immunogenically effective amount” is an amount of the expressedpolypeptide that is sufficient to evoke an active immune responseagainst the polypeptide in the subject to which the pharmaceuticalformulation is administered. In particular embodiments, the dosage issufficient to produce a protective immune response (as defined above).The degree of protection conferred need not be complete or permanent, aslong as the benefits of administering the immunogenic polypeptideoutweigh any disadvantages thereof.

Thus, the present invention provides a method of administering a nucleicacid to a cell, the method comprising contacting the cell with the virusvector, virus particle and/or composition of this invention.

A further aspect of the invention is a method of administering the virusvector, virus particle and/or virus capsid of this invention to asubject. Thus, the present invention also provides a method ofdelivering a nucleic acid to a subject, comprising administering to thesubject a virus particle, virus vector and/or composition of thisinvention. Administration of the virus vectors, virus particles and/orcapsids according to the present invention to a human subject or ananimal in need thereof can be by any means known in the art. Optionally,the virus vector, virus particle and/or capsid is delivered in atreatment effective or prevention effective dose in a pharmaceuticallyacceptable carrier.

The virus vectors and/or capsids of the invention can further beadministered to elicit an immunogenic response (e.g., as a vaccine).Typically, immunogenic compositions of the present invention comprise animmunogenically effective amount of virus vector and/or capsid incombination with a pharmaceutically acceptable carrier. Optionally, thedosage is sufficient to produce a protective immune response (as definedabove). The degree of protection conferred need not be complete orpermanent, as long as the benefits of administering the immunogenicpolypeptide outweigh any disadvantages thereof. Subjects and immunogensare as described above.

Dosages of the virus vector and/or capsid to be administered to asubject depend upon the mode of administration, the disease or conditionto be treated and/or prevented, the individual subject's condition, theparticular virus vector or capsid, and the nucleic acid to be delivered,and the like, and can be determined in a routine manner. Exemplary dosesfor achieving therapeutic effects are titers of at least about 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵ transducing units,optionally about 10⁸-10¹³ transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal, in utero(or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular [including administration to skeletal, diaphragm and/orcardiac muscle], intradermal, intrapleural, intracerebral, andintraarticular), topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intralymphatic, and the like, as well as direct tissue or organinjection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmmuscle or brain). Administration can also be to a tumor (e.g., in ornear a tumor or a lymph node). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand/or prevented and on the nature of the particular vector that isbeing used.

Administration to skeletal muscle according to the present inventionincludes but is not limited to administration to skeletal muscle in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/ordigits. Suitable skeletal muscles include but are not limited toabductor digiti minimi (in the hand), abductor digiti minimi (in thefoot), abductor hallucis, abductor ossis metatarsi quinti, abductorpollicis brevis, abductor pollicis longus, adductor brevis, adductorhallucis, adductor longus, adductor magnus, adductor pollicis, anconeus,anterior scalene, articularis genus, biceps brachii, biceps femoris,brachialis, brachioradialis, buccinator, coracobrachialis, corrugatorsupercilii, deltoid, depressor anguli oris, depressor labii inferioris,digastric, dorsal interossei (in the hand), dorsal interossei (in thefoot), extensor carpi radialis brevis, extensor carpi radialis longus,extensor carpi ulnaris, extensor digiti minimi, extensor digitorum,extensor digitorum brevis, extensor digitorum longus, extensor hallucisbrevis, extensor hallucis longus, extensor indicis, extensor pollicisbrevis, extensor pollicis longus, flexor carpi radialis, flexor carpiulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimibrevis (in the foot), flexor digitorum brevis, flexor digitorum longus,flexor digitorum profundus, flexor digitorum superficialis, flexorhallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexorpollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus,gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis,iliocostalis lumborum, iliocostalis thoracis, illiacus, inferiorgemellus, inferior oblique, inferior rectus, infraspinatus,interspinalis, intertransversi, lateral pterygoid, lateral rectus,latissimus dorsi, levator anguli oris, levator labii superioris, levatorlabii superioris alaeque nasi, levator palpebrae superioris, levatorscapulae, long rotators, longissimus capitis, longissimus cervicis,longissimus thoracis, longus capitis, longus colli, lumbricals (in thehand), lumbricals (in the foot), masseter, medial pterygoid, medialrectus, middle scalene, multifidus, mylohyoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus, occipitalis, omohyoid, opponens digiti minimi, opponenspollicis, orbicularis oculi, orbicularis oris, palmar interossei,palmaris brevis, palmaris longus, pectineus, pectoralis major,pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius,piriformis, plantar interossei, plantaris, platysma, popliteus,posterior scalene, pronator quadratus, pronator teres, psoas major,quadratus femoris, quadratus plantae, rectus capitis anterior, rectuscapitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intermedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor,and any other suitable skeletal muscle as known in the art.

The virus vector and/or capsid can be delivered to skeletal muscle byintravenous administration, intra-arterial administration,intraperitoneal administration, limb perfusion, (optionally, isolatedlimb perfusion of a leg and/or arm; see, e.g. Arruda et al., (2005)Blood 105: 3458-3464), and/or direct intramuscular injection. Inparticular embodiments, the virus vector and/or capsid is administeredto a limb (arm and/or leg) of a subject (e.g., a subject with musculardystrophy such as DMD) by limb perfusion, optionally isolated limbperfusion (e.g., by intravenous or intra-articular administration). Inembodiments of the invention, the virus vectors and/or capsids of theinvention can advantageously be administered without employing“hydrodynamic” techniques. Tissue delivery (e.g., to muscle) of priorart vectors is often enhanced by hydrodynamic techniques (e.g.,intravenous/intravenous administration in a large volume), whichincrease pressure in the vasculature and facilitate the ability of thevector to cross the endothelial cell barrier. In particular embodiments,the viral vectors and/or capsids of the invention can be administered inthe absence of hydrodynamic techniques such as high volume infusionsand/or elevated intravascular pressure (e.g., greater than normalsystolic pressure, for example, less than or equal to a 5%, 10%, 15%,20%, 25% increase in intravascular pressure over normal systolicpressure). Such methods may reduce or avoid the side effects associatedwith hydrodynamic techniques such as edema, nerve damage and/orcompartment syndrome.

Administration to cardiac muscle includes administration to the leftatrium, right atrium, left ventricle, right ventricle and/or septum. Thevirus vector and/or capsid can be delivered to cardiac muscle byintravenous administration, intra-arterial administration such asintra-aortic administration, direct cardiac injection (e.g., into leftatrium, right atrium, left ventricle, right ventricle), and/or coronaryartery perfusion.

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration.

Delivery to a target tissue can also be achieved by delivering a depotcomprising the virus vector and/or capsid. In representativeembodiments, a depot comprising the virus vector and/or capsid isimplanted into skeletal, cardiac and/or diaphragm muscle tissue or thetissue can be contacted with a film or other matrix comprising the virusvector and/or capsid. Such implantable matrices or substrates aredescribed in U.S. Pat. No. 7,201,898.

In particular embodiments, a virus vector and/or virus capsid accordingto the present invention is administered to skeletal muscle, diaphragmmuscle and/or cardiac muscle (e.g., to treat and/or prevent musculardystrophy, heart disease [for example, PAD or congestive heartfailure]).

In representative embodiments, the invention is used to treat and/orprevent disorders of skeletal, cardiac and/or diaphragm muscle.

In a representative embodiment, the invention provides a method oftreating and/or preventing muscular dystrophy in a subject in needthereof, the method comprising: administering a treatment or preventioneffective amount of a virus vector of the invention to a mammaliansubject, wherein the virus vector comprises a heterologous nucleic acidencoding dystrophin, a mini-dystrophin, a micro-dystrophin, myostatinpropeptide, follistatin, activin type II soluble receptor, IGF-1,anti-inflammatory polypeptides such as the Ikappa B dominant mutant,sarcospan, utrophin, a micro-dystrophin, laminin-α2, α-sarcoglycan,β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, an antibody orantibody fragment against myostatin or myostatin propeptide, and/or RNAiagainst myostatin. In particular embodiments, the virus vector can beadministered to skeletal, diaphragm and/or cardiac muscle as describedelsewhere herein.

Alternatively, the invention can be practiced to deliver a nucleic acidto skeletal, cardiac or diaphragm muscle, which is used as a platformfor production of a polypeptide (e.g., an enzyme) or functional RNA(e.g., RNAi, microRNA, antisense RNA) that normally circulates in theblood or for systemic delivery to other tissues to treat and/or preventa disorder (e.g., a metabolic disorder, such as diabetes [e.g.,insulin], hemophilia [e.g., Factor IX or Factor VIII], amucopolysaccharide disorder [e.g., Sly syndrome, Hurler Syndrome, ScheieSyndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo SyndromeA, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.] or alysosomal storage disorder such as Gaucher's disease[glucocerebrosidase] or Fabry disease [α-galactosidase A] or a glycogenstorage disorder such as Pompe disease [lysosomal acid α glucosidase]).Other suitable proteins for treating and/or preventing metabolicdisorders are described herein. The use of muscle as a platform toexpress a nucleic acid of interest is described in U.S. Patentpublication US 2002/0192189.

Thus, as one aspect, the invention further encompasses a method oftreating and/or preventing a metabolic disorder in a subject in needthereof, the method comprising: administering a treatment or preventioneffective amount of a virus vector of the invention to skeletal muscleof a subject, wherein the virus vector comprises a heterologous nucleicacid encoding a polypeptide, wherein the metabolic disorder is a resultof a deficiency and/or defect in the polypeptide. Illustrative metabolicdisorders and heterologous nucleic acids encoding polypeptides aredescribed herein. Optionally, the polypeptide is secreted (e.g., apolypeptide that is a secreted polypeptide in its native state or thathas been engineered to be secreted, for example, by operable associationwith a secretory signal sequence as is known in the art). Without beinglimited by any particular theory of the invention, according to thisembodiment, administration to the skeletal muscle can result insecretion of the polypeptide into the systemic circulation and deliveryto target tissue(s). Methods of delivering virus vectors to skeletalmuscle is described in more detail herein.

The invention can also be practiced to produce antisense RNA, RNAi orother functional RNA (e.g., a ribozyme) for systemic delivery.

The invention also provides a method of treating and/or preventingcongenital heart failure or PAD in a subject in need thereof, the methodcomprising administering a treatment or prevention effective amount of avirus vector of the invention to a mammalian subject, wherein the virusvector comprises a heterologous nucleic acid encoding, for example, asarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), an angiogenic factor,phosphatase inhibitor I (I-1) and fragments thereof (e.g., I1C), RNAiagainst phospholamban; a phospholamban inhibitory or dominant-negativemolecule such as phospholamban S16E, a zinc finger protein thatregulates the phospholamban gene, β2-adrenergic receptor, β2-adrenergicreceptor kinase (BARK), PI3 kinase, calsarcan, a β-adrenergic receptorkinase inhibitor (βARKct), inhibitor 1 of protein phosphatase 1 andfragments thereof (e.g., I1C), S100A1, parvalbumin, adenylyl cyclasetype 6, a molecule that effects G-protein coupled receptor kinase type 2knockdown such as a truncated constitutively active bARKct, Pim-1,PGC-1α, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1,mir-133, mir-206, mir-208 and/or mir-26a.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector and/or virus capsids of theinvention in a local rather than systemic manner, for example, in adepot or sustained-release formulation. Further, the virus vector and/orvirus capsid can be delivered adhered to a surgically implantable matrix(e.g., as described in U.S. Patent Publication No. US-2004-0013645-A1).

The virus vectors and/or virus capsids disclosed herein can beadministered to the lungs of a subject by any suitable means, optionallyby administering an aerosol suspension of respirable particles comprisedof the virus vectors and/or virus capsids, which the subject inhales.The respirable particles can be liquid or solid. Aerosols of liquidparticles comprising the virus vectors and/or virus capsids may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer, as is known to those of skill inthe art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particlescomprising the virus vectors and/or capsids may likewise be producedwith any solid particulate medicament aerosol generator, by techniquesknown in the pharmaceutical art.

The virus vectors and virus capsids can be administered to tissues ofthe CNS (e.g., brain, eye) and may advantageously result in broaderdistribution of the virus vector or capsid than would be observed in theabsence of the present invention.

In particular embodiments, the delivery vectors of the invention may beadministered to treat diseases of the CNS, including genetic disorders,neurodegenerative disorders, psychiatric disorders and tumors.Illustrative diseases of the CNS include, but are not limited toAlzheimer's disease, Parkinson's disease, Huntington's disease, Canavandisease, Leigh's disease, Refsum disease, Tourette syndrome, primarylateral sclerosis, amyotrophic lateral sclerosis, progressive muscularatrophy, Pick's disease, muscular dystrophy, multiple sclerosis,myasthenia gravis, Binswanger's disease, trauma due to spinal cord orhead injury, Tay Sachs disease, Lesch-Nyan disease, epilepsy, cerebralinfarcts, psychiatric disorders including mood disorders (e.g.,depression, bipolar affective disorder, persistent affective disorder,secondary mood disorder), schizophrenia, drug dependency (e.g.,alcoholism and other substance dependencies), neuroses (e.g., anxiety,obsessional disorder, somatoform disorder, dissociative disorder, grief,post-partum depression), psychosis (e.g., hallucinations and delusions),dementia, paranoia, attention deficit disorder, psychosexual disorders,sleeping disorders, pain disorders, eating or weight disorders (e.g.,obesity, cachexia, anorexia nervosa, and bulemia) and cancers and tumors(e.g., pituitary tumors) of the CNS.

Disorders of the CNS include ophthalmic disorders involving the retina,posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabeticretinopathy and other retinal degenerative diseases, uveitis,age-related macular degeneration, glaucoma).

Most, if not all, ophthalmic diseases and disorders are associated withone or more of three types of indications: (1) angiogenesis, (2)inflammation, and (3) degeneration. The delivery vectors of the presentinvention can be employed to deliver anti-angiogenic factors;anti-inflammatory factors; factors that retard cell degeneration,promote cell sparing, or promote cell growth and combinations of theforegoing.

Diabetic retinopathy, for example, is characterized by angiogenesis.Diabetic retinopathy can be treated by delivering one or moreanti-angiogenic factors either intraocularly (e.g., in the vitreous) orperiocularly (e.g., in the sub-Tenon's region). One or more neurotrophicfactors may also be co-delivered, either intraocularly (e.g.,intravitreally) or periocularly.

Uveitis involves inflammation. One or more anti-inflammatory factors canbe administered by intraocular (e.g., vitreous or anterior chamber)administration of a delivery vector of the invention.

Retinitis pigmentosa, by comparison, is characterized by retinaldegeneration. In representative embodiments, retinitis pigmentosa can betreated by intraocular (e.g., vitreal administration) of a deliveryvector encoding one or more neurotrophic factors.

Age-related macular degeneration involves both angiogenesis and retinaldegeneration. This disorder can be treated by administering theinventive deliver vectors encoding one or more neurotrophic factorsintraocularly (e.g., vitreous) and/or one or more anti-angiogenicfactors intraocularly or periocularly (e.g., in the sub-Tenon's region).

Glaucoma is characterized by increased ocular pressure and loss ofretinal ganglion cells. Treatments for glaucoma include administrationof one or more neuroprotective agents that protect cells fromexcitotoxic damage using the inventive delivery vectors. Such agentsinclude N-methyl-D-aspartate (NMDA) antagonists, cytokines, andneurotrophic factors, delivered intraocularly, optionallyintravitreally.

In other embodiments, the present invention may be used to treatseizures, e.g., to reduce the onset, incidence or severity of seizures.The efficacy of a therapeutic treatment for seizures can be assessed bybehavioral (e.g., shaking, ticks of the eye or mouth) and/orelectrographic means (most seizures have signature electrographicabnormalities). Thus, the invention can also be used to treat epilepsy,which is marked by multiple seizures over time.

In one representative embodiment, somatostatin (or an active fragmentthereof) is administered to the brain using a delivery vector of theinvention to treat a pituitary tumor. According to this embodiment, thedelivery vector encoding somatostatin (or an active fragment thereof) isadministered by microinfusion into the pituitary. Likewise, suchtreatment can be used to treat acromegaly (abnormal growth hormonesecretion from the pituitary). The nucleic acid (e.g., GenBank AccessionNo. J00306) and amino acid (e.g., GenBank Accession No. P01166; containsprocessed active peptides somatostatin-28 and somatostatin-14) sequencesof somatostatins are known in the art.

In particular embodiments, the vector can comprise a secretory signal asdescribed in U.S. Pat. No. 7,071,172.

In representative embodiments of the invention, the virus vector and/orvirus capsid is administered to the CNS (e.g., to the brain or to theeye). The virus vector and/or capsid may be introduced into the spinalcord, brainstem (medulla oblongata, pons), midbrain (hypothalamus,thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland),cerebellum, telencephalon (corpus striatum, cerebrum including theoccipital, temporal, parietal and frontal lobes. cortex, basal ganglia,hippocampus and portaamygdala), limbic system, neocortex, corpusstriatum, cerebrum, and inferior colliculus. The virus vector and/orcapsid may also be administered to different regions of the eye such asthe retina, cornea and/or optic nerve.

The virus vector and/or capsid may be delivered into the cerebrospinalfluid (e.g., by lumbar puncture) for more disperse administration of thedelivery vector. The virus vector and/or capsid may further beadministered intravascularly to the CNS in situations in which theblood-brain barrier has been perturbed (e.g., brain tumor or cerebralinfarct).

The virus vector and/or capsid can be administered to the desiredregion(s) of the CNS by any route known in the art, including but notlimited to, intrathecal, intra-ocular, intracerebral, intraventricular,intravenous (e.g., in the presence of a sugar such as mannitol),intranasal, intra-aural, intra-ocular (e.g., intra-vitreous,sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon'sregion) delivery as well as intramuscular delivery with retrogradedelivery to motor neurons.

In particular embodiments, the virus vector and/or capsid isadministered in a liquid formulation by direct injection (e.g.,stereotactic injection) to the desired region or compartment in the CNS.In other embodiments, the virus vector and/or capsid may be provided bytopical application to the desired region or by intra-nasaladministration of an aerosol formulation. Administration to the eye, maybe by topical application of liquid droplets. As a further alternative,the virus vector and/or capsid may be administered as a solid,slow-release formulation (see, e.g., U.S. Pat. No. 7,201,898).

In yet additional embodiments, the virus vector can used for retrogradetransport to treat and/or prevent diseases and disorders involving motorneurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscularatrophy (SMA), etc.). For example, the virus vector can be delivered tomuscle tissue from which it can migrate into neurons.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

EXAMPLES Example 1. Combinatorial Engineering and Selection ofAntibody-Evading AAV Vectors (AAV1e Clones 1-26)

The method for generating antibody evading AAVe mutants is as follows. Ageneral schematic description of the approach is provided in FIG. 1. Asan example, the first step involves identification of conformational 3Dantigenic epitopes on the AAV capsid surface from cryo-electronmicroscopy. Selected residues within antigenic motifs are then subjectedto mutagenesis using degenerate primers with each codon substituted bynucleotides NNK and gene fragments combined together by Gibson assemblyand/or multistep PCR. Capsid-encoding genes containing a degeneratelibrary of mutated antigenic motifs are cloned into a wild type AAVgenome to replace the original Cap encoding DNA sequence yielding aplasmid library. Plasmid libraries are then transfected into 293producer cell lines to generate AAV capsid libraries, which can then besubjected to selection. Successful generation of AAV libraries isconfirmed via DNA sequencing (FIG. 2). In order to select for new AAVstrains that can escape neutralizing antibodies (NAbs), AAV librariesare subjected to multiple rounds of infection in specific cells ortissues in the presence of a helper virus such as adenovirus with orwithout different monoclonal antibodies, polyclonal antibodies or serumcontaining anti-AAV antibodies. Cell lysates harvested from at least oneround of successful infection and replication are sequenced to identifysingle AAV isolates escaping antibody neutralization.

As a nonlimiting specific example, common antigenic motifs on the AAV1capsid protein (VP1) were subjected to mutagenesis as described above.The degenerate libraries were then subjected to infection in endothelialcells in culture for five cycles of infection and replication. Cellswere infected with AAV1 libraries on day 0, infected with adenovirus atday 1 and cell lysates as well as supernatant were obtained at day 7post-infection for repeating the cycle of infection and replication.This procedure was repeated five times following which, fifteen totwenty isolated clones from each library were subjected to DNA sequenceanalysis (FIG. 2). Each unique sequence was labeled as AAV1e(# number),where the number depicts the specific clonal isolate (Tables 6.1 to6.4).

For validation of AAV1e mutants and their ability to escapeneutralization, AAV1 neutralizing antibodies, 4E4 (FIG. 3 top) and 5H7(FIG. 3 bottom) were serially diluted in DMEM+5% FBS on a 96 well plate.AAV1 and AAV1e clones packaging a CBA-Luc cassette (5e7 vg/well) wereadded and incubated with antibody on a 96 well plate for 30 min at roomtemperature. 293 cells (4e5 cells/well) were added into thevirus+antibody mix and incubated at 37° C., 5% CO₂ incubator for 48 h.Final volume of antibody, virus and cell mixture is 100 ul. Medium wasthen discarded from individual wells and replaced with 25 ul of passivelysis buffer. After 30 min incubation at room temperature, 25 ul ofluciferin was added and reporter transgene expression (transductionefficiency) was assayed using a Victor3 illuminometer.

For validation of AAV1e mutants in mouse models in vivo (FIG. 4), a doseof 1e9 vg/ul was pre-incubated with neutralizing antibodies 4E4 (1:500)or 5H7 (1:10), or with PBS for 1 h at room temperature. Each mouse (6-8weeks old, BALB/c, female) was injected with 20 ul of the virus andantibody mixture into each gastrocnemius muscle in the hind leg (2e10vg/leg) through an intramuscular injection.

Mice were anesthetized with isoflurane and injected with 150 ul ofRediJect D-lucifercin intraperitoneally (IP) at different time intervalsfor live animal imaging and luciferase reporter expression. Luciferaseactivities of each mouse were imaged 1 min after the injection using aXenogen IVIS Lumina® system. Live animal luciferase imaging wasperformed at 1 week and 4 weeks post-injection and luciferase activitiesquantified to determine differences in the ability of AAV1e clones toevade neutralizing antibodies (FIG. 4).

For further enhancement of antibody evading properties, mutationsdiscovered in AAV1e clones were combined on capsids to generate newAAV1e strains (clones 18 through 20). These clones were subjected to invitro transduction assays in order to determine their ability to evadeantibody neutralization. Clones AAV1e18-20 demonstrated the ability toescape both monoclonal antibodies against AAV1 or human serum samplecontaining polyclonal antibodies (FIG. 5).

Example 2. Rational Engineering of Antibody-Evading AAV Vectors (AAV1eSeries 27-36, AAV9e1, and AAV9e2)

Current WT AAV vectors are likely to have pre-existing antibodiestargeted against the capsid surface, which prevents efficienttransduction. Vectors of this invention overcome these limitations.

This invention provides AAV antibody escape variants that retaintransduction efficiency. They are engineered to overcome pre-existingantibody responses based on capsid interaction sites and capsid-antibodystructures, and can be further engineered to target specific tissues.

We have designed AAV1 as well as AAV9 variants to escape anti-AAV capsidmonoclonal binding and host antibody neutralization based on antigenicepitope information attained from 3D structural characterization of AAVcapsids, receptor binding sites, and AAV-antibody complex structuresdetermined by cryo-electron microscopy and image reconstruction. Thesevectors contain amino acid alterations in variable regions of thecapsid, which have been established as common antigenic motifs (CAMs;Table 5). Amino acid residues within these CAMs have been modified togenerate novel AAV strains that can escape neutralizing antibodies (AAVeseries) in order to overcome pre-existing immunity (Tables 7 and 8),which has been reported to be detrimental to AAV transduction efficacyin pre-clinical animal studies and in human clinical trials. We havetested the mutants described herein and observe, using biochemicalapproaches including dot blots and ELISA (FIGS. 6, 7, 9 and 11), thatthese mutants escape recognition by antibodies targeted at the parentalcapsid, escape neutralization in the presence of anti-capsid antibodies(FIGS. 8 and 10), and display significantly reduced recognition by seraobtained from patients participating in a clinical trial utilizing AAV1as the gene delivery vector (FIG. 10).

TABLE 5Representative list of common antigenic motifs (CAMs) found on different AAV serotypes and isolates (respective VP1 numbering ofresidues and different amino acid residues is shown). CAM1 (SEQ ID NO:)CAM3 (SEQ ID NO:) CAM4-1 (SEQ ID NO:) CAM4-2 CAM5 (SEQ ID NO:) AAV1262-SASTGAS-268 (303) 370-VFMIPQYGYL-379 (304) 451-NQSGSAQNK-459 (305)472-SV-473 493-KTDNNNSN-500 (306) AAV2 262-SQSGAS-267 (311)369-VFMVPQYGYL-378 (312) 450-TPSGTTTQS-458 (313) 471-RD-472492-SADNN1SE-499 (314) AAV3 262-SQSGAS-267 (319)369-VFMVPQYGYL-378 (320) 451-TTSGTTNQS-459 (321) 472-SL-473493-ANDNNNSN-500 (322) AAV4 253-RLGESLQS-260 (327)360-VFMVPQYGYC-369 (328) 445-GTTLNAGTA-453 (329) 466-SN-467487-ANQNYKIPATGS-498 (330 AAV5 249-EIKSGSVDGS-258 (335)360-VFTLPQYGYA-369 (336) 440-STNNTGGVQ-448 (337) 458-AN-459479-SG'VNRAS-485 (338) AAV6 262-SASTGAS-268 (343)370-VFMIPQYGYL-379 (344) 45I-NQSGSAQNK-459 (345) 472-SV-473493-KTDNNNSN-500 (346) AAV7 263-SETAGST-269 (351)371-VFMIPQYGYL-380 (352) 453-NPGGTAGNR-461 (353) 474-AE-475495-LDQNNNSN-502 (354) AAV8 263-NGTSGGAT-270 (359)372-VFMIPQYGYL-381 (360) 453-TTGGTANTQ-461 (361) 474-AN-475495-TGQNNNSN-502 (362) AAV9 262-NSTSGGSS-269 (367)371-VFMIPQYGYL-380 (368) 45I-INGSGQNQQ-459 (369) 472-AV-473493-VTQNNNSE-500 (370) AAVrh8 262-NGTSGGST-269 (375)371-VFMVPQYGYL-380 (376) 45I-QTTGTGGTQ-459 (377) 472-AN-473493-TNQNNNSN-500 (378) AAVrh10 263-NGTSGGST-270 (383)372-VFMIPQYGYL-381 (384) 453-STGGTAGTQ-461 (385) 474-SA-475495-LSQNNNSN-502 (386) AAV10 263-NGTSGGST-270 (391)372-VFMIPQYGYL-381 (392) 453-STGGTQGTQ-461 (393) 474-SA-475495-LSQNNNSN-502 (394) AAV11 253-RLGTTSSS-260 (399)360-VFMVPQYGYC-369 (400) 444-GETLNQGNA-452 (401) 465-AF-466486-ASQNYKIPASGG-497 (402) AAV12 262-RIGTTANS-269 (407)369-VFMVPQYGYC-378 (408) 453-GNSLNQGTA-461 (409) 474-AY-475495-ANQNYKIPASGG-506 (410) AAVrh32.33 253-RLGTTSNS-260 (415)360-VFMVPQYGYC-369 (416) 444-GETLNQGNA-452 (417) 465-AF-466486-ASQNYKIPASGG-497 (418) Bovine AAV 255-RLGSSNAS-262 (423)362-VFMVPQYGYC-371 (424) 447-GGTLNQGNS-455 (425) 468-SG-469489-ASQNYKIPQGRN-500 (426) Avian AAV 265-RIQGPSGG-272 (431)375-IYTIPQYGYC-384 (432) 454-VSQAGSSGR-462 (433) 475-AA-476496-ASNITKNNVFSV-507 (434) CAM6 (SEQ ID NO:) CAM7 (SEQ ID NO:)CAM8 (SEQ ID NO:) CAM9-1 CAM9-2 (SEQ ID NO:) AAVI 528-KDDEDKF-534 (307)547-SAGASN-552 (308) 588-STDPATGDVH-597 (309) 709-AN-710716-DNNGLYT-722 (310) AAV2 527-KDDEEKF-533 (315) 546-GSEKTN-551 (316)587-NRQAATADVN-596 (317) 708-VN-709 715-DTNGVYS-721 (318) AAV3528-KDDEEKF-534 (323) 547-GTTASN-552 (324) 588-NTAPTTGTVN-597 (325)709-VN-710 716-DTNGVYS-722 (326) AAV4 527-GPADSKF-533 (331)545-QNGNTA-560 (332) 586-SNLPTVDRLT-595 (333) 707-NS-708714-DAAGKYT-720 (334) AAV5 515-LQGSNTY-521 (339) 534-ANPGTTAT-541 (340)577-TTAPATGTYN-586 (341) 697-QF-698 704-DSTGEYR-710 (342) AAV6528-KDDKDKF-534 (347) 547-SAGASN-552 (348) 588-STDPATGDVH-597 (349)709-AN-710 716-DNNGLYT-722 (350) AAV7 530-KDDEDRF-536 (355)549-GATNKT-554 (356) 589-NTAAQTQVVN-598 (357) 710-TG-711717-DSQGVYS-723 (358) AAV8 530-KDDEERF-536 (363) 549-NAARDN-554 (364)590-NTAPQIGTVNS-600 (365) 711-TS-712 718-NTEGVYS-724 (366) AAV9528-KEGEDRF-534 (371) 547-GTGRDN-552 (372) 588-QAQAQTGWVQ-597 (373)709-NN-710 716-NTEGVYS-722 (374) AAVrh8 528-KDDDDRF-534 (379)547-GAGNDG-552 (380) 588-NTQAQTGLVH-597 (381) 709-TN-710716-NTEGVYS-722 (382) AAVrh10 530-KDDEERF-536 (387) 549-GAGKDN-554 (388)590-NAAPIVGAVN-599 (389) 711-TN-712 718-NTDGTYS-724 (390) AAV10530-KDDEERF-536 (395) 549-GAGRDN-554 (396) 590-NTGPIVGNVN-599 (397)711-TN-712 718-NTEGTYS-724 (398) AAV11 526-GPSDGDF-532 (403)544-VTGNTT-549 (404) 585-TTAPITGNVT-594 (405) 706-SS-707713-DTTGKYT-719 (406) AAV12 535-GAGDSDF-541 (411) 553-PSGNTT-558 (412)594-TTAPHIANLD-603 (413) 715-NS-716 722-DNAGNYH-728 (414) AAVrh32.33526-GPSDGDF-532 (419) 544-VTGNTT-549 (420) 585-TTAPITGNVT-594 (421)706-SS-707 713-DTTGKYT-719 (422) Bovine AAV 529-ANDATDF-535 (427)547-ITGNTT-552 (428) 588-TTVPTVDDVD-597 (429) 709-DS-710716-DNAGAYK-722 (430) Avian AAV 533-FSGEPDR-539 (435)552-VYDQTTAT-559 (436) 595-VTPGTRAAVN-604 (437) 716-AD-717723-SDTGSYS-729 (438)

TABLE 6.1AAV1e1-7. List of novel neutralizing antibody evading AAV1e strainsisolated after screening and selection. Each strain is labeled asAAV1eN, where N is the strain number. Amino acid residues that wereselected by this approach within the different common antigenicmotifs are listed with VPI capsid protein numbering. In each case,15-25 clones isolated from the library screen were sent for sequenceanalysis the relative frequencies of each strain is also listed.Nab Evading Novel amino acid sequence identified AAV1e strainsin corresponding AAV1e isolate Frequency AAV1e1456-QVRG-459 (SEQ ID NO: 22) 10/19 AAV1e2 456-GRGG-459 (SEQ ID NO: 24) 1/19 AAV1e3 456-SGGR-459 (SEQ ID NO: 25)  1/19 AAV1e4456-ERPR-459 (SEQ ID NO: 23)  1/19 AAV1e5 456-SERR-459 (SEQ ID NO: 26) 1/19 AAV1e6 456-LRGG-459 (SEQ ID NO: 27)  1/19 AAV1e7456-ERPR-459 (SEQ ID NO: 23), D595N  4/19

TABLE 6.2 AAV1e8-16. List of novel neutralizing antibody evading AAV1estrains isolated after screening and selection (Cont'd) Nab EvadingNovel amino acid sequence identified AAV1e strainsin corresponding AAV1e isolate Frequency AAV1e8493-PGGNATR-499 (SEQ ID NO: 30) 15/15 AAV1e9588-TADHDTKGVL-597 (SEQ ID NO: 32) 15/24 AAV1e10588-VVDPDKKGVL-597 (SEQ ID NO: 33)  1/24 AAV1e11588-AKDTGPLNVM-597 (SEQ ID NO: 34)  2/24 AAV1e12588-QTDAKDNGVQ-597 (SEQ ID NO: 35)  1/24 AAV1e13588-DKDPWLNDVI-597 (SEQ ID NO: 36)  1/24 AAV1e14588-TRDGSTESVL-597 (SEQ ID NO: 37)  2/24 AAV1e15588-VIDPDQKGVL-597 (SEQ ID NO: 38)  1/24 AAV1e16588-VNDMSNYMVH-597 (SEQ ID NO: 39)  1/24

TABLE 6.3(AAV1e17-20). List of novel neutralizing antibody evading AAV1e generated by makingvarious rationally engineered permutations and combinations of amino acid sequencesderived from AAV1e6, AAV1e8 and AAV1e9. Nab Evading AAV1e strains(Combination mutant strains)Amino acid sequences combined by rational mutagenesis AAV1e17(456-LRGG-459, SEQ ID NO: 27) + (493-PGGNATR-499, SEQ ID NO: 30) AAV1e18(456-LRGG-459, SEQ ID NO: 27) + (588-TADHDTKGVL-597, SEQ ID NO: 32)AAV1e19 (493-PGGNATR-499, SEQ ID NO: 30) + (588-TADHDTKGVL-597, SEQID NO: 32) AAV1e20(456-LRGG-459, SEQ ID NO: 27) + (493-PGGNATR-499, SEQ ID NO: 30) +(588-TADHDTKGVL-597, SEQ ID NO: 32)

TABLE 6.4AAV1e21-26. List of novel neutralizing antibody evading AAV1e strainsisolated after screening and selection (Cont'd.) These novel AAVle strains contain new  sequences listed below in addition to the AAV1e8 sequence493-PGGNATR-499. Briefly, an AAV1e capsid library was generated usingAAV1e8 as the template capsid and randomizing common antigenic motif CAM8(residues 588-597). These were subjected similar screening and isolationprotocols to obtain different novel AAV1e isolates. Nab Evading AAV1eNovel amino strains engineered using acid sequence identified inAAV1e8 as a template corresponding AAVle isolate Frequency AAV1e21588-CNDEMQVQVN-597 (SEQ ID NO: 297) 2/9 AAV1e22588-SPDIVYADVC-597 (SEQ ID NO: 298) 1/9 AAV1e23588-LDDCHNIDVN-597 (SEQ ID NO: 299) 1/9 AAV1e24588-SCDCVTNSVS-597 (SEQ ID NO: 300) 1/9 AAV1e25588-TVDSNPYEVN-597 (SEQ ID NO: 301) 1/9 AAV1e26588-GDDHPNPDVL-597 (SEQ ID NO: 302) 1/9

TABLE 7 AAV1e27-36. List of novel neutralizing antibody evading AAV1estrains generated by making various rationally determined, site-specificmutations on the AAV capsid protein. Single mutants and multiple sitemutants are shown. Nab Evading Site-specific amino acid mutationsgenerated by rational AAV1e strains mutagenesis AAV1e27 S472R AAV1e28V473D AAV1e29 N500E AAV1e30 A456T + Q457T + N458Q + K459S AAV1e31T492S + K493A AAV1e32 S586R + S587G + S588N + T589R AAV1e33 A456T +Q457T + N458Q + K459S + T492S + K493A AAV1e34 A456T + Q457T + N458Q +K459S + S586R + S587G + S588N + T589R AAV1e35 T492S + K493A + S586R +S587G + S588N + T589R AAV1e36 A456T + Q457T + N458Q + K459S + T492S +K493A + S586R + S587G + S588N + T589R

TABLE 8 AAV9e1 & AAV9e2. Proof of concept studies establishing therational design of novel neutralizing antibody evading AAV9e strains.Table lists the different site-specific point mutations made in AAV9 byrational mutagenesis. Antibody Evading Site-specific amino acidmutations generated by AAV1e strains rational mutagenesis AAV9e1 S454V +Q456V AAV9e2 I451Q + G453Q + Q456S + N457A + N459 insertion

Example 3. Structure-Based Iterative Evolution of Antigenically AdvancedAAV Variants for Therapeutic Gene Transfer

Cells, viruses and antibodies. HEK293 and MB114 cells were maintained inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalbovine serum (FBS) (ThermoFisher, Waltham, Mass.), 100 units/ml ofpenicillin and 101±g/ml of streptomycin (P/S) (ThermoFisher, Waltham,Mass.) in 5% CO₂ at 37° C. Murine adenovirus 1 (MAV-1) was purchasedfrom American Type Culture Collection (ATCC, Mannassas, Va.) andamplified by infecting MB114 cells at a multiplicity of infection (MOI)of 1. At day 6 post-infection (approximately 50% cytopathic effect(CPE)), media containing progeny MAV-1 viruses were harvested andcentrifuged at 3000 g for 5 min, and the supernatant stored at −80° C.for subsequent evolution studies. Mouse anti-AAV1 monoclonal antibodiesADK1a, 4E4 and 5H7 have been described previously. De-identified andnaïve human serum samples were purchased from Valley Biomedical,Winchester, Va. Naïve serum from rhesus macaques was from the YerkesNational Primate Center. Antisera against AAV1 capsids, generated byimmunizing rhesus macaques intramuscularly (I.M.) with AAV1 capsids wasfrom the Oregon National Primate Center. All mouse, human and non-humanprimate serum used in this study were heat inactivated at 55° C. for 15min prior to use.

Recombinant AAV Production, Purification and Quantification.

Recombinant AAV vectors were produced by transfecting four 150 mm dishescontaining HEK293 cells at 70-80% confluence using polyethylenimine(PEI) according to the triple plasmid protocol. Recombinant vectorspackaging single stranded genomes encoding firefly luciferase driven bythe chicken beta-actin promoter (ssCBA-Luc) or self-complementary greenfluorescence protein driven by a hybrid chicken beta-actin promoter(scCBh-GFP) were generated using this method. Subsequent steps involvingharvesting of recombinant AAV vectors and downstream purification werecarried out as described previously. Recombinant AAV vector titers weredetermined by quantitative PCR (qPCR) with primers that amplify AAV2inverted terminal repeat (ITR) regions, 5′-AACATGCTACGCAGAGAGGGAGTGG-3′(SEQ ID NO:477), 5′-CATGAGACAAGGAACCCCTAGTGATGGAG-3′ (SEQ ID NO:478).

Structural Modeling and Analysis of AAV Antigenic Footprints.

Antigenic footprints of AAV serotypes 1/6, AAV2, AAV5, AAV8 and AAV9were determined using previously resolved structures of AAV capsidscomplexed with different mouse monoclonal antibodies. To restrictdiversity and maximize efficiency of AAV library generation, only aminoacid residues directly in contact with antibodies were included foranalysis. Contact surface residues on each serotype were either alignedby Clustal Omega software or structurally superimposed using PyMOL(Schrödinger, New York City, N.Y.). Structural alignment revealed thatantibody footprints from multiple serotypes overlap in close proximityto the 3-fold symmetry axis, around the 5-fold pore and at the 2-folddepression. Of these so-called common antigenic motifs (CAMs), wedetermined that 12/18 of the antibodies analyzed have direct contact atthe 3-fold symmetry supporting the notion that this region is a criticalantigenic determinant. For the current study, antigenic footprints forthree distinct monoclonal antibodies (4E4, 5H7 and ADK1a) werevisualized on the AAV1 capsid (PDB ID: 3ng9) and roadmap images weregenerated using the RIVEM program.

Generation of AAV Capsid Libraries.

AAV libraries were engineered through saturation mutagenesis of aminoacid residues within different antigenic footprints associated withdistinct monoclonal antibodies described above. Briefly, for Gibsonassembly, twelve oligos with an average length of 70 nucleotides wereordered from IDT (Coralville, Iowa). Each oligo contains at least 15-20nt overlapping homology to the neighboring oligos. Three oligoscontained degenerate nucleotides (NNK) within genomic regions coding fordifferent antigenic footprints. Plasmid libraries were then generated byin vitro assembly of multiple oligos using High Fidelity Gibson AssemblyMix (NEB, Ipswich, Mass.) according to manufacturer instructions. Theassembled fragments were either PCR amplified for 10 cycles usingPhusion HF (NEB, Ipswich, Mass.) or directly cloned into pTR-AAV1**plasmids between the BspEI and SbfI restriction sites. PlasmidpTR-AAV1** contains genes encoding AAV2 Rep and AAV1 Cap with 2 stopcodons at positions 490 and 491 (AAV1 VP1 numbering) introduced by sitedirected mutagenesis (Agilent, Santa Clara, Calif.). The entireconstruct is flanked by AAV2 inverted terminal repeats (ITRs) to enablepackaging and replication of pseudotyped AAV1 libraries upon helpervirus co-infection. It is noteworthy to mention that the AAV1** capsidgene was incorporated prior to library cloning in order to reduce wildtype AAV1 contamination within the different libraries. Ligationreactions were then concentrated and purified by ethanol precipitation.Purified ligation products were electroporated into DHIOB electroMax(Invitrogen, Carlsbad, Calif.) and directly plated on multiple 5245 mm²bioassay dishes (Corning, Corning, N.Y.) to avoid bias from bacterialsuspension cultures. Plasmid DNA from pTR-AAV1CAM libraries was purifiedfrom pooled colonies grown on LB agar plates using a Maxiprep kit(Invitrogen, Carlsbad, Calif.).

Directed Evolution of Novel AAV CAM Strains.

Equal amounts (15 μg each) of each pTR-AAV1CAM library and the Ad helperplasmid, pXX680, were transfected onto HEK293 cells at 70-80% confluencyon each 150 mm dish using PEI to generate CAM viral libraries. AAV CAMlibraries were purified using standard procedures described earlier.MB114 cells were seeded on a 100 mm tissue culture dish overnight toreach 60-70% confluence before inoculation with AAV CAM libraries at anMOI ranging from 1000-10,000. After 24 h post-transduction, MAV-1 wasadded as helper virus to promote AAV replication. At 6 dayspost-infection with MAV-1 (50% CPE), the supernatant was harvested andDNase I resistant vector genomes were quantified on day 7. Mediacontaining replicating AAV strains and MAV-1 obtained from each round ofinfection were then used as inoculum for each subsequent cycle for atotal of 5 rounds of evolution. Subsequent iterative rounds of evolutionwere carried out in a similar fashion with AAV capsid librariescontaining different permutations and combinations of newly evolvedantigenic footprints.

Identification of Newly Evolved AAV Strains.

To analyze sequence diversity of the parental and evolved AAV CAMlibraries, DNase I resistant vector genomes were isolated from media andamplified by Q5 polymerase for 10-18 cycles (NEB, Ipswich, Mass.) usingprimers, 5′-CCCTACACGACGCTCTTCCGATCTNNNNNeagaact a ggaagt-3′ (SEQ IDNO:479) and5′-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNgccaggtaatgctcccatagc-3′ (SEQ IDNO:480). Illumina MiSeq sequencing adaptor for multiplexing was addedthrough a second round of PCR using Q5 Polymerase with P5 and P7primers. After each round of PCR, the products were purified using aPureLink PCR Micro Kit (ThermoFisher, Waltham, Mass.). Quality of theamplicons was verified using a Bioanalyzer (Agilent), and concentrationsquantified using a Qubit spectrometer (ThermoFisher, Waltham, Mass.).Libraries were then prepared for sequencing with a MiSeq 300 Kit v2,following manufacturer instructions, and sequenced on the MiSeq system(Illumina).

Sequencing Data Analysis.

De-multiplexed reads were analyzed via a custom Perl script. Briefly,raw sequencing files were probed for mutagenized regions of interest,and the frequencies of different nucleotide sequences in this regionwere counted and ranked for each library. Nucleotide sequences were alsotranslated, and these amino acid sequences were similarly counted andranked. Amino acid sequence frequencies across libraries were thenplotted in R.

Isolation of AAV CAM Variants for Characterization.

To characterize selected clones from each library, DNase I resistantvector genomes were isolated from media and amplified by Phusion HF(NEB, Ipswich, Mass.) using primers flanking the BspEI and SbfI sites.The PCR products were gel purified, sub-cloned into TOPO cloning vectors(ThermoFisher, Waltham, Mass.) and sent out for standard Sangersequencing (Eton Bioscience, San Diego, Calif.). Unique sequences weresub-cloned into an AAV helper plasmid backbone, pXR, using BspEI andSbfI sites. Unique recombinant AAV CAM variants were produced followinga standard rAAV production protocol as described above.

In Vitro Antibody and Serum Neutralization Assays.

Twenty-five microliters of antibodies or antisera (as specified forindividual experiments) was mixed with an equal volume containingrecombinant AAV vectors (MOI 1,000-10,000) in tissue culture treated,black, glass bottom 96 well plates (Corning, Corning, N.Y.) andincubated at room temperature (RT°) for 30 min. A total of 5×10⁴ HEK293cells in 50 μl of media was then added to each well and the platesincubated in 5% CO₂ at 37° C. for 48 h. Cells were then lysed with 25 μlof 1× passive lysis buffer (Promega, Madison, Wis.) for 30 min at RT.Luciferase activity was measured on a Victor 3 multilabel plate reader(Perkin Elmer, Waltham, Mass.) immediately after addition of 25 μl ofluciferin (Promega, Madison, Wis.). All read outs were normalized tocontrols with no antibody/antisera treatment. Recombinant AAV vectorspackaging ssCBA-Luc transgenes and pre diluted in DMEM+5% FBS+P/S wereutilized in this assay.

In Vive Antibody Neutralization Assay.

Each hind limb of 6-8 week old female BAlb/c mice (Jackson Laboratory,Bay Harbor, Me.) was injected intramuscularly (I.M.) with 2×10¹⁰ AAVpackaging CBA-Luc pre-mixed with three different monoclonal antibodies,4E4, 5H7 and ADK1a, at 1:500, 1:50 and 1:5 dilutions, respectively, in afinal volume of 20 μl. After 4 wk post-injection, luciferase activitywas measured using a Xenogen IVIS Lumina system (PerkinElmer LifeSciences/Caliper Life Sciences, Waltham, Mass.) at 5 minpost-intraperitoneal (I.P.) injection of 175 μl of in vivo D-luciferin(120 mg/kg Nanolight, Pinetop, Ariz.) per mouse. Luciferase activity wasmeasured as photons/sec/cm²/sr and analyzed using Living Image 3.2software (Caliper Life Sciences, Waltham, Mass.).

Generation of Anti-AAV1 Mouse Serum by Immunization.

1×10¹⁰ vg of wtAAV1 in 20 μl of PBS was injected intramuscularly intoeach hind leg of 6-8 week old, female Balb/c mice. Whole blood wascollected by cardiac puncture at 4 wk post-injection and serum wasisolated using standard coagulation and centrifugation protocols.Briefly, mouse blood was coagulated at RT for 30 min and centrifuged at2000 g for 10 min at 4° C. All serum was heat-inactivated at 55° C. for15 min and stored at −80° C.

In Vivo Characterization of AAV CAM Variants in Mice.

A dose of 1×10¹¹ vg of AAV vectors packaging the scCBh-GFP transgenecassette in 200 μl of PBS was injected into C57/B16 mice intravenously(I.V.) via the tail vein. Mice were sacrificed after 3 wk post-injectionand perfused with 4% paraformaldehyde (PFA) in PBS. Multiple organs,including heart, brain, liver and kidney, were harvested. Tissues weresectioned to 50 μm thin slices by vibratome VT1200S (Leica, Welzlar,Germany) and stained for GFP with standard immunohistochemistry3,3′-Diaminobenzidine (DAB) stain procedures described previously. Atleast 3 sections per organ from 3 different mice were submitted forslide scanning. For bio-distribution analysis, 1×10¹¹ vg of AAV vectorspackaging ssCBA-Luc were injected I.V. as mentioned above in Balb/Cmice. After 2 wk post-injection, mice were sacrificed and perfused with1×PBS. Multiple organs, including heart, brain, lung, liver, spleen,kidney and muscle, were harvested. DNA was harvested using DNeasy kit(Qiagen, Hilden, Germany) according to the manufacturer's instructions.Vector genome copy numbers were determined by quantitative PCR (qPCR)using as described previously using luciferase transgene primers,5′-CCTTCGCITCAAAAAATGGAAC-3′ (SEQ ID NO:481), and5′-AAAAGCACTCTGATTGACAAATAC-3′ (SEQ ID NO:482). Viral genome copynumbers were normalized to mouse genomic DNA in each sample. Tissuesamples were also processed for luciferase activity assays byhomogenization in 1×PLB (Promega, Madison, Wis.) using a QiagenTissueLyserII at a frequency of 20 hz for three 45 s pulses. Thehomogenate was spun down, and 20 μl of supernatant mixed with 50 μl ofluciferin (Promega, Madison, Wis.) and immediately measured using aVictor 3 multilabel plate reader (Perkin Elmer, Waltham, Mass.).

Intracerebroventricular (I.C.V.) Injections.

Postnatal day 0 (P0) C57/B16 pups which were anesthetized on ice for 2minutes followed by stereotaxic I.C.V. injections with AAV vectorspackaging the scCBh-GFP transgene cassette. A dose of 3×10⁹ vg in 3 μlof PBS was injected into the left lateral ventricle using a Hamilton 700series syringe with a 26 s gauge needle (Sigma-Aldrich, St. Louis, Mo.),attached to a KOPF-900 small animal stereotaxic instrument (KOPFinstruments, Tujunga, Calif.). All neonatal injections were performed0.5 mm relative to the sagittal sinus, 2 mm rostral to transverse sinusand 1.5 mm deep. After vector administration, mice were revived under aheat lamp and rubbed in the bedding before being placed back with thedam. Mouse brains were harvested at 2 wk post vector administrations(P14). Brains were post fixed and immunostained as described previously.

Western Blots and Electron Microscopy.

A total of 5×10⁹ viral genomes were re-suspended in NuPAGE LDS samplebuffer (Invitrogen, Carlsbad, Calif.). +50 mM 1,4-Dithiothreitol (DTT).Samples were ran on NuPAGE 4-12% Bis-Tris Gel and transferred ontopolyvinylidene fluoride (PVDF) membrane. (Invitrogen, Carlsbad, Calif.).AAV capsid proteins were detected using mouse monoclonal antibody B1(1:50) and secondary goat anti-mouse conjugated to horseradishperoxidase (HRP) (Jackson Immuno Research Labs, West Grove, Pa.). For EMstudies, 1×10⁹ vg/l of virus was prepared in PBS and absorbed on aFormvar/Carbon 400 mesh, Cu grid (TED Pella, Redding, Calif.). Sampleswere negative stained with 2% uranyl acetate and analyzed using a ZeissSupra 25 field emission scanning electron microscope.

Structural Analysis of AAV-Antibody Complexes Enables an IterativeApproach to Evolve Novel AAV Variants.

We analyzed previously resolved, cryo-reconstructed structures of AAV1capsids complexed with four different fragment antigen binding (Fab)regions of anti-AAV1 monoclonal antibodies. Three-dimensionalreconstruction revealed that this subset of antibodies nearly masks theentire AAV1 capsid surface (FIG. 12A). We then identified a subset ofcapsid surface residues (through construction of roadmap images) thatlie within these antigenic footprints and are implicated in directcontact with the different antibodies (FIG. 12B). Further analysis andcomparison with different AAV serotypes revealed a prominent clusteringof common antigenic footprints at the 3-fold symmetry axis on the capsidsurface. Specifically, amino acid residues within three surface regions,common antigenic motif 4 (CAM4; 456-AQNK-459, SEQ ID NO:483), commonantigenic motif 5 (CAM5; 493-KTDNNNS-499, SEQ ID NO:484) and commonantigenic motif 8 (CAM8; 588-STDPATGDVH-597, SEQ ID NO:309) wereselected for saturation mutagenesis and generation of different AAVlibraries. It is important to note that the different CAMs listed aboveare subsets of variable regions (VRs) 4, 5 and 8 outlined previously.Each AAV capsid library was then subjected to five rounds of directedevolution in vascular endothelial cells, which are highly permissive tothe parental AAV1 strain (FIG. 12C). Novel AAV variants were identifiedand combination AAV libraries were engineered using the latter astemplates. Iterative rounds of evolution and capsid engineering yieldednovel antigenically advanced AAV strains characterized in the currentstudy (FIG. 12D).

Antigenic Footprints on the AAV Capsid Surface are Remarkably Plasticand Evolvable.

As outlined above, the AAV CAM4, CAM5, and CAM8 libraries were subjectedto 5 rounds of directed evolution. Libraries were then sequenced usingthe MiSeq system (Illumina), wherein each unselected (parental) librarywas sequenced at ˜2×10⁶ reads and selected (evolved) libraries sequencedat ˜2×10⁵ reads. De-multiplexed reads were probed for mutagenizedregions of interest with a custom Perl script, with a high percentage ofreads mapping to these regions for all libraries (FIGS. 20-21). At boththe nucleotide and amino acid level, all unselected librariesdemonstrated high diversity and minimal bias towards any particularsequence, while evolved libraries showed dramatic enhancement inrepresentation of one or more lead variants (FIGS. 13A-C, E-G). Further,within the top ten selected variants for each library, many amino acidsequences showed similarities at multiple residues (FIGS. 13E-G). Forinstance, in the evolved CAM5 library 97.55% of sequences spanning themutagenized region of interest read TPGGNATR (SEQ ID NO:485), whileminor variants largely mimicked this sequence (FIG. 13F). In case ofCAM8, we observed significant enrichment (86.6%) for a variant withamino acid residues TADHDTKGV (SEQ ID NO:486) (FIG. 13G). The evolvedCAM4 library demonstrated higher plasticity (QVRG (SEQ ID NO:22),69.57%; ERPR (SEQ ID NO:23), 14.05%; SGGR (SEQ ID NO:25), 3.62%) asevidenced by the range of amino acid residues tolerated within thatantigenic region (FIG. 13A). We then generated a combination AAV library(CAM58, FIG. 13D), which carries the lead epitope from the evolved CAM5library and a randomized CAM8 region. Interestingly, subjecting thislibrary to directed evolution yielded the wild type AAV1 sequence in theCAM8 region (92.27%), i.e., STDPATGDVH (SEQ ID NO:309) (FIG. 13H).Although a secondary variant with the sequence DLDPKATEVE (SEQ IDNO:487)was also enriched (1.4%) (FIG. 13D), the latter observation demonstratesthe evolutionary and structural constraints imposed by the interactionbetween CAM5 and CAM8 regions. These constraints were further evaluatedby rational combination of different epitopes derived from these novelCAM4, 5 or 8 variants. Nevertheless, these results corroborate thenotion that antigenic footprints on the AAV capsid surface are mutableand can be evolved into novel footprints, while maintaining infectivity.

Individually Evolved AAV CAM Variants are Similar to the Parental AAV1Serotype.

Multiple, evolved AAV variants were selected from each library forsubsequent characterization, specifically, CAM101-107 (region 4), CAM108(region 5) and CAM109-116 (region 8). All CAM variants packaging thessCBA-Luc genome were produced and their transduction efficienciesassessed in vascular endothelial cells (FIGS. 19A-19C). A single CAMvariant from each evolved library that displayed the highesttransduction efficiency was shortlisted for further characterization.Specifically, CAM106 (456-SERR-459, SEQ ID NO:26), CAM108(492-TPGGNATR-499, SEQ ID NO:485) and CAM109 (588-TADHDTKGVL-597, SEQ IDNO:32)) showed similar to modestly improved transduction efficiencycompared to parental AAV1 on vascular endothelial cells. Theseobservations support the notion that antigenic footprints can bere-engineered and evolved, while maintaining or improving upon theendogenous attributes of the corresponding parental AAV strain. Furtherevaluation of the physical properties of these lead CAM variantsconfirmed that yield (vector genome titers), capsid morphology (EM), andpackaging efficiency (proportion of full-to-empty particles) werecomparable to parental AAV1 vectors (FIGS. 19A-19C).

Individual CAM Variants Evade Neutralization by Monoclonal Antibodies.

We first evaluated the ability of single region CAM variants to escapeneutralization by mouse monoclonal antibodies, ADK1a, 4E4 and 5H7described previously. As shown in FIGS. 14A-C, each CAM variant shows adistinct NAb escape profile. As expected, parental AAV1 was neutralizedby all MAbs tested at different dilutions. The CAM106 and CAM108variants were resistant to neutralization by 4E4, while CAM109 wascompletely neutralized similar to AAV1 (FIG. 14A). Next, we determinedthat CAM108 and CAM109 both escape neutralization by 5H7, whereas CAM106was significantly affected by 5H7 similar to AAV1 (FIG. 14B). WithADK1a, CAM106 was completely resistant to neutralization, while CAM108and CAM109 were both effectively neutralized (FIG. 14C).

In Vivo Neutralization Profile of CAM Variants Against MonoclonalAntibodies.

To further test whether the ability of CAM variants to escapeneutralization can be reproduced in vivo, AAV1 and CAM variantspackaging ssCBA-Luc were mixed with the corresponding MAbs and injectedintramuscularly into mice. In the absence of MAbs, all CAM variants andAAV1 showed similar luciferase transgene expression in mouse muscle(FIG. 14E). In the presence of antibodies, the neutralization profilesof the CAM variants corroborated results from in vitro studies. Briefly,CAM106 was resistant to ADK1a and 4E4, while CAM108 efficientlytransduces mouse muscle in the presence of 4E4 or 5H7 and CAM109 evades5H7 with high efficiency. Importantly, AAV1 transduction of mouse musclewas completely abolished when co-administered with any of theseantibodies (FIGS. 14F-H). Quantitative analysis of luciferase transgeneexpression by CAM variants normalized to AAV1 confirmed theseobservations (FIG. 14I).

Iterative Engineering of Complex Antigenic Footprints on Single RegionCAM Variants.

Based on promising results from MAb neutralization studies, wehypothesized that combining different, evolved antigenic footprints willallow better NAb evasion. To achieve such, we generated four variantsthrough a combination of rational mutagenesis, library generation anditerative evolution. First, we observed that rational combination ofantigenic footprints from CAM106 and CAM108 yielded a functional andstable AAV variant, dubbed CAM117 (FIG. 15A). However, we observed thatamino acid residues constituting antigenically advanced footprints onCAM108 and CAM109 were not structurally compatible (reduced viral titer)In order to facilitate structural compatibility, we generated a new AAVcapsid library using CAM108 as a template and by carrying out saturationmutagenesis of amino acid residues in region 8. After 3 iterative cyclesof directed evolution on vascular endothelial cells, several viablevariants were generated (FIG. 15A). After initial characterization,CAM125 (region 5, 492-TPGGNATR-499 (SEQ ID NO:485); region 8,588-DLDPKATEVE-597 (SEQ ID NO:487)) was selected for further analysis.We then iteratively engineered a third variant (CAM130) by grafting theevolved antigenic footprint from CAM106 onto CAM125. The CAM130 variantcontains the following amino acid residues in three distinct antigenicfootprints—region 4, 456-SERR-459 (SEQ ID NO:26; region 5,492-TPGGNATR-499 (SEQ ID NO:485) and region 8, 588-DLDPKATEVE-597 (SEQID NO:487) (FIG. 15A). All three iteratively engineered variants,CAM117, CAM125 and CAM130 show similar physical attributes compared toparental AAV1 with regard to titer and proportion of full-to-emptyparticles (FIGS. 19A-19C).

CAM117, CAM 125 and CAM130 Escape Neutralizing Antisera fromPre-Immunized Mice.

To test whether antigenically advanced CAM variants can demonstrateescape from polyclonal neutralizing antibodies found in serum, wesero-converted mice by immunization with wild type AAV1 capsids.Overall, while antisera obtained from individual mice efficientlyneutralized AAV1, CAM117, CAM125 and CAM130 display increased resistanceto neutralization (FIGS. 15B-D). Briefly, we tested antisera dilutionsranging over two orders of magnitude (1:3200 to 1:50) to generatesigmoidal neutralization curves. As seen in FIGS. 15B-D, when comparedto AAV1, the CAM variants show a dramatic shift to the right indicatingimproved ability to evade anti-AAV1 serum. In particular, the serumconcentration required for 50% neutralization of transduction (ND₅₀) issignificantly higher in case of each CAM variant compared to parentalAAV1 in each individual subject (FIGS. 15B-D). Furthermore, we observedan incremental ability to evade NAbs with each iterativeengineering/evolution step. Specifically, the most antigenicallyadvanced variant, CAM130 displays a 8-16 fold improvement in ND₅₀ values(FIGS. 15B-D). These results corroborate the notion that antigenicfootprints on AAV capsid are modular and cumulative in their ability tomediate NAb evasion. A similar, but less robust trend was observed withregard to the neutralizing potential of serum obtained from naïve miceas control (FIG. 15E).

CAM130 Efficiently Evades Neutralization by Non-Human Primate Antisera.

To validate whether our approach can be translated in larger animalmodels, we tested the ability of AAV1 and the lead variant, CAM130 toevade NAbs generated in non-human primates. Briefly, we subjected AAVvectors to neutralization assays using serum collected at threedifferent time points—pre-immunization (naïve), 4 wks and 9 wkspost-immunization. All macaques sero-converted after immunization withNAb titers at the highest levels in week 4 and declining at week 9 insubjects 1 and 2, and increased potency at week 9 in subject 3 (FIGS.16A-I). Moreover, naïve sera from subjects 1 and 3 prior to immunizationwere able to neutralize AAV1 effectively (FIGS. 16A and 16G). We testedantisera dilutions ranging over two orders of magnitude (1:320 to 1:5)to generate neutralization curves as described earlier. Antiseraobtained at 4 wks after immunization neutralized AAV1 effectively atND₅₀>1:320. In contrast, CAM130 displayed a significant shift to theright and improved resistance to neutralization compared to AAV1 by 4-16fold (FIGS. 16B, 16E, 16H). A similar trend and enhancement inresistance to NAbs was observed in the case of CAM130 when evaluatingantisera obtained at 9 wks post-immunization (FIGS. 16C, 16F, 16I).Further, these results strongly support the notion that antigenicity ofAAV capsids can be re-engineered to escape broadly neutralizingantibodies from different animal species on the basis of structural cuesobtained from mouse MAb footprints.

CAM130 Efficiently Evades NAbs in Primate and Human Sera.

To test whether CAM130 can evade NAbs in the general non-human primateand human population, we tested serum samples obtained from a cohort of10 subjects each. We evaluated a fixed serum dilution of 1:5 to reflectcurrently mandated exclusion criteria employed in ongoing clinicaltrials for hemophilia and other indications requiring systemic AAVadministration. As seen in FIG. 17A, primate subjects p-A and p-Bdisplayed high NAb titers that completely neutralized both AAV1 andCAM130. At the other end of the spectrum, subjects p-I and p-J showed nopre-existing immunity to AAV capsids and did not effectively neutralizeAAV1 or CAM130. However, serum samples for subjects p-C through p-Hefficiently neutralized AAV1 and reduced transduction efficiency below50% of untreated controls. In contrast, serum samples p-C through p-Hwere unable to neutralize the antigenically advanced CAM130 variant.Thus, CAM130 shows exceptional NAb evasion in this cohort by evading 8out of 10 serum samples (FIG. 17A). We then utilized a similar approachto test serum from 10 human subjects. Using clinically relevantexclusion criteria (1:5 dilution), we segregated the human sera into twohigh titer (h-A and h-B), six intermediate titer (h-C through h-H) andtwo modest titer sub-groups that neutralized AAV1 effectively.Strikingly, CAM130 was able to evade polyclonal NAbs in human sera for8/10 samples tested (FIG. 17B). Taken together, these studies stronglysupport the notion that the antigenically advanced CAM130 variant cansignificantly expand the patient cohort.

CAM130 Displays a Favorable Transduction Profile In Vivo.

We compared the in vivo tissue tropism, transduction efficiency andbiodistribution of CAM130 to the parental AAV1 strain in mice. A dose of1×10¹¹ vg/mouse of AAV vectors packaging scCBh-GFP was injectedintravenously into 6-8 week old female BALB/c mice via the tail vein. At2 wks post injection, CAM130 showed an enhanced cardiac GFP expressionprofile compared to AAV1, while differences in the liver wereunremarkable. In particular, more GFP-positive cardiac myofibers aredetectable in CAM130 treated animals compared to the AAV1 cohort. Wethen administered 1×10¹¹ vg/mouse of AAV vectors packaging ssCBA-Lucgenomes intravenously as described above. In contrast to GFP expressionfrom self-complementary CAM130 vectors, no significant differences werenoted in luciferase activity within the heart for ssCAM130 vs.AAV1-treated mice (FIG. 18A). However, a modest, albeit statisticallyinsignificant increase in luciferase expression was observed within theliver (FIG. 18C). Transduction efficiencies in other major organs, i.e.,lung, brain, kidney and spleen, were low. Importantly, no differenceswere noted in the systemic biodistribution of CAM130 and AAV1 vectors.Consistent with earlier reports, ˜10-fold higher vector genome copynumbers were detected in the liver compared to cardiac tissue for bothCAM130 and AAV1 vectors (FIGS. 18B and 18D).

To further compare the potency and tropism of CAM130 to AAV1, weevaluated the transduction profiles of the latter two strains followingCNS administration. A dose of 3×10⁹ vg/mouse of AAV1 or CAM130 packagingscCBh-GFP genomes was injected by intra-CSF administration in neonatalmice. Both AAV1 and CAM130 spread well within the brain with a generalpreference for transducing the ipsilateral side more readily than thecontralateral hemisphere. Similar to cardiac tissue, a greater number ofGFP-positive cells are observed in the case of CAM130 compared to AAV1.In particular, CAM130 appears to transduce a greater number of neurons,particularly within the motor cortex, cortex and most prominently in thehippocampus. The potential mechanism(s) for the improved transductionprofile displayed by CAM130 in cardiac and CNS tissue could potentiallyarise from post-entry trafficking events that are currently underinvestigation. More importantly, these in vivo results confirm thatantigenic footprints on AAV capsids can be engineered to effectivelyevade NAbs, while simultaneously controlling cellular/tissue tropism aswell as biodistribution profile and improving potency.

Similarly, AAV1e mutants demonstrate robust and neuron-specific geneexpression in the brain following intracranial administration. DifferentAAV1e vectors packaging an scGFP expression cassette were administeredinto the cerebrospinal fluid of P0 mice by stereotaxic injection intolateral ventricles. The vector dose administered was 3×10⁹ vectorgenomes/animal. Mice were sacrificed at 3 weeks post-injection andbrains processed using DAB immunohistochemistry and image reporter geneexpression in cerebellum, olfactory bulb, cortex and hippocampus.

Example 4: AAV8e Antibody Evading Mutants

Evolved mutants AAV8e01, AAV8e04 and AAV8e05 demonstrate improvedtransduction in comparison with parental AAV8 isolate in humanhepatocarcinoma cells (Huh7). Briefly, cells were incubated withdifferent AAV8 derived variants at 10,000 vector genomes per cell for 24hours. Quantitation of luciferase transgene activity revealed over 2 logincrease in transduction efficiency of AAV8e01 over AAV8 (dotted line);over 1 log order increase for AAV8e05 and ˜2-fold increase in the caseof AAV8e04. These results corroborate the generation of novel AAV8variants that demonstrate robust transduction of transformed humanhepatocytes in culture compared to the state-of-the-art natural isolate.These results are shown in FIG. 22.

AAV8e mutants demonstrate the ability to escape neutralization by mousemonoclonal antibodies generated specifically against AAV8. Briefly,human hepatocarcinoma cells were incubated with different AAV8e mutantsor wild type (WT) AAV8 vectors packaging luciferase transgene cassetteswith or without neutralizing antibodies. Each monoclonal antibody (mAb)was directed against different antigenic epitopes located on the AAV8capsid surface. As shown in FIGS. 23A-23C, AAV8e04 and AAV8e05 escapeneutralization by mAbs HL2381 (FIG. 23A), HL2383 (FIG. 23B) and ADK8(FIG. 23C) tested at different dilutions. In contrast, the parental AAV8strain is neutralized effectively under these conditions.

Nonlimiting examples of AAV8e mutants of this invention are listed inTable 9.

TABLE 9 AAV8e mutants Name Clone Sequence Description AAV8e01 CAM84a455-SNGRGV-460 (SEQ ID NO: 488) Single 8CAM-4a AAV8e02 CAM84b455-VNTSING-461 (SEQ ID NO: 489) Single 8CAM-4b AAV8e03 CAM84c455-IRGAGAV-461 (SEQ ID NO: 490) Single 8CAM-4c AAV8e04 CAM85a494-YPGGNYK-501 (SEQ ID NO: 491) Single 8CAM-5a AAV8e05 CAM88a586-KQKNVN-591 (SEQ ID NO: 492) Single 8CAM-8a AAV8e06 CAM88b586-RMSSIK-591 (SEQ ID NO: 493) Single 8CAM-8b AAV8e07 CAM845a455-SNGRGV-460 (SEQ ID NO: 488) + Double 8CAM-4a-5a494-YPGGNYK-501 (SEQ ID NO: 491) AAV8e08 CAM848a455-SNGRGV-460 (SEQ ID NO:488) + Double 8CAM-4a-8a586-KQKNVN-591 (SEQ ID NO: 492)

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

Sequences

SEQUENCESAAV1 capsid protein (GenBank Accession No. AAD27757) (SEQ ID NO: 1)   1MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD  61KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121AKKRVLEPLG LVEEGAKTAP GKKRPVEQSP QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE 181SVPDPQPLGE PPATPAAVGP TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI 241TTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301INNNWGFRPK RLNFKLENIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQ 361GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEEVP 421FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP 481GPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD EDKFFPMSGV 541MIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG 601ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA 661EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGL 721YTEPRPIGTR YLTRPLAAV2 capsid protein (GenBank Accession No. YP_680426) (SEQ ID NO: 2)   1MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD  61KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 121AKKRVLEPLG LVEEPVKTAP GKKRPVEHSP VEPDSSSGTG KAGQQPARKR LNFGQTGDAD 181SVPDPQPLGQ PPAAPSGLGT NTMATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI 241TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301NNNWGFRPKR LNFKLENIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG 361CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFT FSYTFEDVPF 421HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT PSGTTTQSRL QFSQAGASDI RDQSRNWLPG 481PCYRQQRVSK TSADNNNSEY SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL 541IFGKQGSEKT NVDIEKVMIT DEEEIRTTNP VATEQYGSVS TNLQRGNRQA ATADVNTQGV 601LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN TPVPANPSTT 661FSAAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNPEIQY TSNYNKSVNV DFTVDTNGVY 721SEPRPIGTRY LTRNLAAV3 capsid protein (GenBank Accession No. AAC55049) (SEQ ID NO: 3)   1MAADGYLPDW LEDNLSEGIR EWWALKPGVP QPKANQQHQD NRRGLVLPGY KYLGPGNGLD  61KGEPVNEADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLQEDTSF GGNLGRAVFQ 121AKKRILEPLG LVEEAAKTAP GKKGAVDQSP QEPDSSSGVG KSGKQPARKR LNFGQTGDSE 181SVPDPQPLGE PPAAPTSLGS NTMASGGGAP MADNNEGADG VGNSSGNWHC DSQWLGDRVI 241TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301NNNWGFRPKK LSFKLFNIQV RGVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG 361CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFQ FSYTFEDVPF 421HSSYAHSQSL DRLMNPLIDQ YLYYLNRTQG TTSGTTNQSR LLFSQAGPQS MSLQARNWLP 481GPCYRQQRLS KTANDNNNSN FPWTAASKYH LNGRDSLVNP GPAMASHKDD EEKFFPMHGN 541LIFGKEGTTA SNAELDNVMI TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTGTVNHQG 601ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT 661TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VDFTVDTNGV 721YSEPRPIGTR YLTRNLAAV4 capsid protein (GenBank Accession No. NP_044927) (SEQ ID NO: 4)   1MTDGYLPDWL EDNLSEGVRE WWALQPGAPK PKANQQHQDN ARGLVLPGYK YLGPGNGLDK  61GEPVNAADAA ALEHDKAYDQ QLKAGDNPYL KYNHADAEFQ QRLQGDTSFG GNLGRAVFQA 121KKRVLEPLGL VEQAGETAPG KKRPLIESPQ QPDSSTGIGK KGKQPAKKKL VFEDETGAGD 181GPPEGSTSGA MSDDSEMRAA AGGAAVEGGQ GADGVGNASG DWHCDSTWSE GHVTTTSTRT 241WVLPTYNNHL YKRLGESLQS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGMRPK 301AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE GSLPPFPNDV 361FMVPQYGYCG LVTGNTSQQQ TDRNAFYCLE YFPSQMLRTG NNFEITYSFE KVPFHSMYAH 421SQSLDRLMNP LIDQYLWGLQ STTTGTTLNA GTATTNFTKL RPTNFSNFKK NWLPGPSIKQ 481QGFSKTANQN YKIPATGSDS LIKYETHSTL DGRWSALTPG PPMATAGPAD SKFSNSQLIF 541AGPKQNGNTA TVPGTLIFTS EEELAATNAT DTDMWGNLPG GDQSNSNLPT VDRLTALGAV 601PGMVWQNRDI YYQGPIWAKI PHTDGHFHPS PLIGGFGLKH PPPQIFIKNT PVPANPATTF 661SSTPVNSFIT QYSTGQVSVQ IDWEIQKERS KRWNPEVQFT SNYGQQNSLL WAPDAAGKYT 721EPRAIGTRYL THHLAAV5 capsid protein (GenBank Accession No. AAD13756) (SEQ ID NO: 5)   1MSFVDHPPDW LEEVGEGLRE FLGLEAGPPK PKPNQQHQDQ ARGLVLPGYN YLGPGNGLDR  61GEPVNRADEV AREHDISYNE QLEAGDNPYL KYNHADAEFQ EKLADDTSFG GNLGKAVFQA 121KKRVLEPFGL VEEGAKTAPT GKRIDDHFPK RKKARTEEDS KPSTSSDAEA GPSGSQQLQI 181PAQPASSLGA DTMSAGGGGP LGDNNQGADG VGNASGDWHC DSTWMGDRVV TKSTRTWVLP 241SYNNHQYREI KSGSVDGSNA NAYFGYSTPW GYFDFNRFHS HWSPRDWQRL INNYWGFRPR 301SLRVK1FNIQ VKEVTVQDST TTIANNLTST VQVFTDDDYQ LPYVVGNGTE GCLPAFPPQV 361FTLPQYGYAT LNRDNTENPT ERSSFFCLEY FPSKMLRTGN NFEFTYNFEE VPFHSSFAPS 421QNLFKLANPL VDQYLYRFVS TNNTGGVQFN KNLAGRYANT YKNWFPGPMG RTQGWNLGSG 481VNRASVSAFA TTNRMELEGA SYQVPPQPNG MTNNLQGSNT YALENTMIFN SQPANPGTTA 541TYLEGNMLIT SESETQPVNR VAYNVGGQMA TNNQSSTTAP ATGTYNLQEI VPGSVWMERD 601VYLQGPIWAK IPETGAHFHP SPAMGGFOLK HPPPMMLIKN TPVPGNITSF SDVPVSSFIT 661QYSTGQVTVE MEWELKKENS KRWNPEIQYT NNYNDPQFVD FAPDSTGEYR TTRPIGTRYL 721TRPL AAV6 capsid protein (GenBank Accession No. AAB95450) (SEQ ID NO: 6)  1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ121 AKKRVLEPFG LVEEGAKTAP GKKRPVEQSP QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE181 SVPDPQPLGE PPATPAAVGP TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI241 TTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQ361 GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEDVP421 FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP481 GPCYRQQPVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGV541 MIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA661 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGL721 YTEPRPIGTR YLTRPLAAV7 capsid protein (GenBank Accession No. AAN03855) (SEQ ID NO: 7)   1MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD NGRGLVLPGY KYLGPFNGLD  61KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121AKKRVLEPLG LVEEGAKTAP AKKRPVEPSP QRSPDSSTGI GKKGQQPARK RLNFGQTGDS 181ESVPDPQPLG EPPAAPSSVG SGTVAAGGGA PMADNNEGAD GVGNASGNWH CDSTWLGDRV 241ITTSTRTWAL PTYNNHLYKQ ISSETAGSTN DNTYFGYSTP WGYFDFNRFH CHFSPRDWQR 301LINNNWGFRP KKLRFKLFNI QVKEVTTNDG VTTIANNLTS TIQVFSDSEY QLPYVLGSAH 361QGCLPPFPAD VFMIPQYGYL TLNNGSQSVG RSSFYCLEYF PSQMLRTGNN FEFSYSFEDV 421PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNPGGTAGN RELQFYQGGP STMAEQAKNW 481LPGPCFRQQR VSKTLDQNNN SNFAWTGATK YHLNGRNSLV NPGVAMATHK DDEDRFFPSS 541GVLIFGKTGA TNKTTLENVL MTNEEEIRPT NPVATEEYGI VSSNLQAANT AAQTQVVNNQ 601GALPGMVWQN RDVYLQGPIW AKIPHTDGNF HPSPLMGGFG LKHPPPQILI KNTPVPANPP 661EVFTPAKFAS FITQYSTGQV SVEIEWELQK ENSKRWNPEI QYTSNFEKQT GVDFAVDSQG 721VYSEPRPIGT RYLTRNLAAV8 capsid protein (GenBank Accession No. AAN03857) (SEQ ID NO: 8)   1MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD  61KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPARK RLNFGQTGDS 181ESVPDPQPLG EPPAAPSGVG PNTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV 241ITTSTRTWAL PTYNNHLYKQ ISNGTSGGAT NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA 361HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFQFTYTFED 421VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQTTGGTANT QTLGESQGGP NTMANQAKNW 481LPGPCYRQQR VSTTTGQNNN SNFAWTAGTK YHLNGRNSLA NPGIAMATHK DDEERFFPSN 541GILIFGKQNA ARDNADYSDV MLTSEEEIKT TNPVATEEYG IVADNLQQQN TAPQIGTVNS 601QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP 661PTTFNQSKLN SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TSVDFAVNTE 721GVYSEPRPIG TRYLTRNLAAV9 capsid protein (GenBank Accession No. AAS99264) (SEQ ID NO: 9)   1MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD  61KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 121AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE 181SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI 241TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR 301LINNNWGFRP KRLNFKLENT QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH 361EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV 421PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP 481GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS 541LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG 601ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT 661AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV 721YSEPRPIGTR YLTRNLAAVrh.8 capsid protein (GenBank Accession No. AA088183) (SEQ ID NO: 10)  1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ121 AKKRVLEPLG LVEEGAKTAP GKKRPVEQSP QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE181 SVPDPQPLGE PPAAPSGLGP NTMASGGGAP MADNNEGADG VGNSSGNWHC DSTWLGDRVI241 TTSTRTWALP TYNNHLYKQI SNGTSGGSTN DNTYFGYSTP WGYFDFNRFH CHFSPRDWQR301 LINNNWGFRP KRLNFKLFNI QVKEVTTNEG TKTIANNLTS TVQVFTDSEY QLPYVLGSAH361 QGCLPPFPAD VFMVPQYGYL TLNNGSQALG RSSFYCLEYF PSQMLRTGNN FQFSYTFEDV421 PFHSSYAHSQ SLDRLMNPLI DQYLYYLVRT QTTGTGGTQT LAFSQAGPSS MANQARNWVP481 GPCYRQQRVS TTTNQNNNSN FAWTGAAKFK LNGRDSLMNP GVAMASHKDD DDRFFPSSGV541 LIFGKQGAGN DGVDYSQVLI TDEEEIKATN PVATEEYGAV AINNQAANTQ AQTGLVHNQG601 VIPGMVWQNR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK NTPVPADPPL661 TFNQAKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSTN VDFAVNTEGV721 YSEPRPIGTR YLTRNLAAVrh.10 capsid protein (GenBank Accession No. AA088201) (SEQ ID NO: 11)  1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ121 AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS181 ESVPDPQPIG EPPAGPSGLG SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV241 ITTSTRTWAL PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ301 RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA361 HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFEFSYQFED421 VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQSTGGTAGT QQLLFSQAGP NNMSAQAKNW481 LPGPCYRQQR VSTTLSQNNN SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS541 GVLMFGKQGA GKDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQQN AAPIVGAVNS601 QGALPGMVWQ NRDVYLQGPT WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP661 PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TNVDFAVNTD721 GTYSEPRPIG TRYLTRNLAAV10 capsid protein (GenBank Accession No. AAT46337) (SEQ ID NO: 12)  1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ121 AKKRVLEPLG LVEEAAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPAKK RLNFGQTGES181 ESVPDPQPIG EPPAGPSGLG SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV241 ITTSTRTWAL PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ301 RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKT1ANNLT STIQVFTDSE YQLPYVLGSA361 HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFEFSYTFED421 VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQSTGGTQGT QQLLFSQAGP ANMSAQAKNW481 LPGPCYRQQR VSTTLSQNNN SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS541 GVLMFGKQGA GRDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQAN TGPIVGNVNS601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP661 PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TNVDFAVNTE721 GTYSEPRPIG TRYLTRNLAAV11 capsid protein (GenBank Accession No. AAT46339) (SEQ ID NO: 13)  1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ121 AKKRVLEPLG LVEEGAKTAP GKKRPLESPQ EPDSSSGIGK KGKQPARKRL NFEEDTGAGD181 GPPEGSDTSA MSSDIEMRAA PGGNAVDAGQ GSDGVGNASG DWHCDSTWSE GKVTTTSTRT241 WVLPTYNNHL YLRLGTTSSS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGLRPK301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE GSLPPFPNDV361 FMVPQYGYCG IVTGENQNQT DRNAFYCLEY FPSQMLRTGN NFEMAYNFEK VPFHSMYAHS421 QSLDRLMNPL LDQYLWHLQS TTSGETLNQG NAATTFGKIR SGDFAFYRKN WLPGPCVKQQ481 RFSKTASQNY KIPASGGNAL LKYDTHYTLN NRWSNIAPGP PMATAGPSDG DFSNAQLIFP541 GPSVTGNTTT SANNLLFTSE EEIAATNPRD TDMFGQIADN NQNATTAPIT GNVTAMGVLP601 GMVWQNRDIY YQGPIWAKIP HADGHFHPSP LIGGFGLKHP PPQIFIKNTP VPANPATTFT661 AARVDSFITQ YSTGQVAVQI EWEIEKERSK RWNPEVQFTS NYGNQSSMLW APDTTGKYTE721 PRVIGSRYLT NHLAAV12 capsid protein (GenBank Accession No. ABI16639) (SEQ ID NO: 14)  1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NGRGLVLPGY KYLGPFNGLD 61 KGEPVNEADA AALEHDKAYD KQLEQGDNPY LKYNHADAEF QQRLATDTSF GGNLGRAVFQ121 AKKRILEPLG LVEEGVKTAP GKKRPLEKTP NRPTNPDSGK APAKKKQKDG EPADSARRTL181 DFEDSGAGDG PPEGSSSGEM SHDAEMRAAP GGNAVEAGQG ADGVGNASGD WHCDSTWSEG241 RVTTTSTRTW VLPTYNNHLY LRIGTTANSN TYNGFSTPWG YFDFNRFHCH FSPRDWQRLI301 NNNWGLRPKS MRVKIFNIQV KEVTTSNGET TVANNLTSTV QIFADSTYEL PYVMDAGQEG361 SFPPFPNDVF MVPQYGYCGV VTGKNQNQTD RNAFYCLEYF PSQMLRTGNN FEVSYQFEKV421 PFHSMYAHSQ SLDRMMNPLL DQYLWHLQST TTGNSLNQGT ATTTYGKITT GDFAYYRKNW481 LPGACIKQQK FSKNANQNYK IPASGGDALL KYDTHTTLNG RWSNMAPGPP MATAGAGDSD541 FSNSQLIFAG PNPSGNTTTS SNNLLFTSEE EIATTNPRDT DMFGQIADNN QNATTAPHIA601 NLDAMGIVPG MVWQNRDIYY QGPIWAKVPH TDGHFHPSPL MGGFGLKHPP PQIFIKNTPV661 PANPNTTFSA ARINSFLTQY STGQVAVQID WEIQKEHSKR WNPEVQFTSN YGTQNSMLWA721 PDNAGNYHEL RAIGSRFLTH HLAAVih.32.33 capsid protein (GenBank Accession No. ACB55318) (SEQ ID NO: 15)  1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ121 AKKRVLEPLG LVEEGAKTAP GKKRPLESPQ EPDSSSGIGK KGKQPAKKRL NFEEDTGAGD181 GPPEGSDTSA MSSDIEMRAA PGGNAVDAGQ GSDGVGNASG DWHCDSTWSE GKVTTTSTRT241 WVLPTYNNHL YLRLGTTSNS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGLRPK301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE GSLPPFPNDV361 FMVPQYGYCG IVTGENQNQT DRNAFYCLEY FPSQMLRTGN NFEMAYNFEK VPFHSMYAHS421 QSLDRLMNPL LDQYLWHLQS TTSGETLNQG NAATTFGKIR SGDFAFYRKN WLPGPCVKQQ481 RFSKTASQNY KIPASGGNAL LKYDTHYTLN NRWSNIAPGP PMATAGPSDG DFSNAQLIFP541 GPSVTGNTTT SANNLLFTSE EEIAATNPRD TDMFGQIADN NQNATTAPIT GNVTAMGVLP601 GMVWQNRDIY YQGPIWAKIP HADGHFHPSP LIGGFGLKHP PPQIFIKNTP VPANPATTFT661 AARVDSFITQ YSTGQVAVQI EWEIEKERSK RWNPEVQFTS NYGNQSSMLW APDTTGKYTE721 PRVIGSRYLT NHLBovine AAV capsid protein (GenBank Accession No. YP_024971) (SEQ ID NO: 16)  1 MSFVDHPPDW LESIODGFRE FLGLEAGPPK PKANQQKQDN ARGLVLPGYK YLGPGNGLDK 61 GDPVNFADEV AREHDLSYQK QLEAGDNPYL KYNHADAEFQ EKLASDTSFG GNLGKAVFQA121 KKRILEPLGL VETPDKTAPA AKKRPLEQSP QEPDSSSGVG KKGKQPARKR LNFDDEPGAG181 DGPPPEGPSS GAMSTETEMR AAAGGNGGDA GQGAEGVGNA SGDWHCDSTW SESHVTTTST241 RTWVLPTYNN HLYLRLGSSN ASDTFNGFST PWGYFDFNRF HCHFSPRDWQ RLINNHWGLR301 PKSMQVRIFN IQVKEVTTSN GETTVSNNLT STVQIFADST YELPYVMDAG QEGSLPPFPN361 DVFMVPQYGY CGLVTGGSSQ NQTDRNAFYC LEYFPSQMLR TGNNFEMVYK FENVPFHSMY421 AHSQSLDRLM NPLLDQYLWE LQSTTSGGTL NQGNSATNFA KLTKTNFSGY RKNWLPGPMM481 KQQRFSKTAS QNYKIPQGRN NSLLHYETRT TLDGRWSNFA PGTAMATAAN DATDFSQAQL541 IFAGPNITGN TTTDANNLMF TSEDELRATN PRDTDLFGHL ATNQQNATTV PTVDDVDGVG601 VYPGMVWQDR DIYYQGPIWA KIPHTDGHFH PSPLIGGFGL KSPPPQIFIK NTPVPANPAT661 TFSPARINSF ITQYSTGQVA VKIEWEIQKE RSKRWNPEVQ FTSNYGAQDS LLWAPDNAGA721 YKEPRAIGSR YLTNHLAvian AAV ATCC VR-865 capsid protein (GenBank Accession No. NP_852781) (SEQ IDNO: 17)   1MSLISDAIPD WLERLVKKGV NAAADFYHLE SGPPRPKANQ QTQESLEKDD SRGLVFPGYN  61YLGPFNGLDK GEPVNEADAA ALEHDKAYDL EIKDGHNPYF EYNEADRRFQ ERLKDDTSFG 121GNLGKAIFQA KKRVLEPFGL VEDSKTAPTG DKRKGEDEPR LPDTSSQTPK KNKKPRKERP 181SGGAEDPGEG TSSNAGAAAP ASSVGSSIMA EGGGGPVGDA GQGADGVGNS SGNWHCDSQW 241LENGVVTRTT RTWVLPSYNN HLYKRIQGPS GGDNNNKFFG FSTPWGYFDY NRFHCHFSPR 301DWQRLINNNW GIRPKAMRFR LFNIQVKEVT VQDFNTTIGN NLTSTVQVFA DKDYQLPYVL 361GSATEGTFPP FPADIYTIPQ YGYCTLNYNN EAVDRSAFYC LDYFPSDMLR TGNNFEFTYT 421FEDVPFHSMF AHNQTLDRLM NPLVDQYLWA FSSVSQAGSS GRALHYSRAT KTNMAAQYRN 481WLPGPFFRDQ QIFTGASNIT KNNVFSVWEK GKQWELDNRT NLMQPGPAAA TTFSGEPDRQ 541AMQNTLAFSR TVYDQTTATT DRNQILITNE DEIRPTNSVG IDAWGAVPTN NQSIVTPGTR 601AAVNNQGALP GMVWQNRDIY PTGTHLAKIP DTDNHFHPSP LIGRFGCKHP PPQIFIKNTP 661VPANPSETFQ TAKVASFINQ YSTGQCTVEI FWELKKETSK RWNPEIQFTS NFGNAADIQF 721AVSDTGSYSE PRPIGTRYLT KPL

That which is claimed is:
 1. A recombinant adeno associated virus (AAV)vector, the vector comprising a capsid protein and a nucleic acidencapsidated by the capsid protein; wherein the nucleic acid encodes aheterologous polypeptide or heterologous RNA sequence; and wherein thecapsid protein comprises one or more of the following substitutions,wherein the amino acids are numbered according to the amino acidsequence of SEQ ID NO:8: a) a substitution of the amino acidscorresponding to amino acids 455-460 with the sequence SNGRGV (SEQ IDNO:488), a substitution of the amino acids corresponding to amino acids455-461 with the sequence VNTSING (SEQ ID NO:489), or a substitution ofthe amino acids corresponding to amino acids 455-461 with the sequenceIRGAGAV (SEQ ID NO:490); b) a substitution of the amino acidscorresponding to amino acids 494-501 with the sequence YPGGNYK (SEQ IDNO:491); and/or c) a substitution of the amino acids corresponding toamino acids 586-591 with the sequence KQKNVN (SEQ ID NO:492) or RMSSIK(SEQ ID NO:493).
 2. The recombinant AAV vector of claim 1, wherein theone or more substitutions inhibit neutralization of infectivity of theAAV vector and/or inhibit binding of an antibody to the AAV vector,wherein the antibody binds to a capsid protein comprising the amino acidsequence of SEQ ID NO:8.
 3. The recombinant AAV vector of claim 2,wherein the antibody is selected from the group consisting of HL2381,HL3283, and ADK8.
 4. The recombinant AAV vector of claim 3, wherein theantibody is HL2381.
 5. The recombinant AAV vector of claim 3, whereinthe antibody is HL2383.
 6. The recombinant AAV vector of claim 3,wherein the antibody is ADK8.
 7. The recombinant AAV vector of claim 1,wherein the capsid protein further comprises a substitution at aminoacid 453, 454, 502, 592, 593, 594, 595, 596, 597, 598, 599 and/or 600,in any combination.
 8. The recombinant AAV vector of claim 1, whereinthe capsid protein comprises a substitution of amino acids 455-460 withthe sequence SNGRGV (SEQ ID NO:488).
 9. The recombinant AAV vector ofclaim 1, wherein the capsid protein comprises a substitution of aminoacids 455-460 with the sequence SNGRGV (SEQ ID NO:488) and asubstitution of amino acids 494-501 with the sequence YPGGNYK (SEQ IDNO:491).
 10. The recombinant AAV vector of claim 1, wherein the capsidprotein comprises a substitution of amino acids 455-460 with thesequence SNGRGV (SEQ ID NO:488) and a substitution of amino acids494-501 with the sequence KQKNVN (SEQ ID NO:492).
 11. The recombinantAAV vector claim 1, wherein the heterologous polypeptide is atherapeutic polypeptide.
 12. The recombinant AAV vector of claim 1,wherein the heterologous RNA is a functional RNA.
 13. The recombinantAAV vector of claim 1, wherein the nucleic acid comprises terminalrepeat (TR) sequences from AAV2 or AAV4.
 14. A recombinantadeno-associated virus (AAV) vector, the vector comprising a capsidprotein and a nucleic acid encapsidated by the capsid protein; whereinthe nucleic acid encodes a heterologous polypeptide or heterologous RNAsequence; and wherein the capsid protein comprises one or more of thefollowing substitutions, wherein the amino acids are numbered accordingto the amino acid sequence of SEQ ID NO:8: a) a substitution of theamino acids corresponding to amino acids 455-460 with the sequenceSNGRGV (SEQ ID NO:488), a substitution of the amino acids correspondingto amino acids 455-461 with the sequence VNTSING (SEQ ID NO:489), or asubstitution of the amino acids corresponding to amino acids 455-461with the sequence IRGAGAV (SEQ ID NO:490); b) a substitution of theamino acids corresponding to amino acids 494-501 with the sequenceYPGGNYK (SEQ ID NO:491); and/or c) a substitution of the amino acidscorresponding to amino acids 586-591 with the sequence KQKNVN (SEQ IDNO:492) or RMSSIK (SEQ ID NO:493), and wherein the capsid protein withthe substitutions of one or more of a)-c) is at least about 90%identical to the amino acid sequence of SEQ ID NO:8.
 15. The recombinantAAV vector of claim 14, wherein the capsid protein comprises a sequenceat least about 95% identical to the amino acid sequence of SEQ ID NO:8.16. The recombinant AAV vector of claim 14, wherein the capsid proteincomprises a sequence at least about 97% identical to the amino acidsequence of SEQ ID NO:8.
 17. The recombinant AAV vector of claim 14,wherein the capsid protein comprises a sequence at least about 98%identical to the amino acid sequence of SEQ ID NO:8.
 18. The recombinantAAV vector of claim 14, wherein the capsid protein comprises a sequenceat least about 99% identical to the amino acid sequence of SEQ ID NO:8.19. The recombinant AAV vector of claim 14, wherein the one or moresubstitutions inhibit neutralization of infectivity of the AAV vectorand/or inhibit binding of an antibody to the AAV vector, wherein theantibody binds to a capsid protein comprising the amino acid sequence ofSEQ ID NO:8.
 20. The recombinant AAV vector of claim 19, wherein theantibody is selected from the group consisting of HL2381, HL2383, andADK8.
 21. The recombinant AAV vector of claim 20, wherein the antibodyis HL2381.
 22. The recombinant AAV vector of claim 20, wherein theantibody is HL2383.
 23. The recombinant AAV vector of claim 20, whereinthe antibody is ADK8.
 24. The recombinant AAV vector of claim 14,wherein the capsid protein comprises one or more substitutions at theamino acids corresponding to amino acids 453-461 and 495-502 of SEQ IDNO:8.
 25. The recombinant AAV vector of claim 14, wherein the capsidprotein comprises one or more substitutions at the amino acidscorresponding to amino acids 453-461 and 590-600 of SEQ ID NO:8.
 26. Therecombinant AAV vector of claim 14, wherein the capsid protein comprisesone or more substitutions at the amino acids corresponding to aminoacids 495-502 and 590-600 of SEQ ID NO:8.
 27. The recombinant AAV vectorof claim 14, wherein the capsid protein comprises one or moresubstitutions at the amino acids corresponding to amino acids 453-461,495-502 and 590-600 of SEQ ID NO:8.
 28. The recombinant AAV vector ofclaim 14, wherein the capsid protein comprises a substitution of aminoacids 455-460 of SEQ ID NO:8 with the sequence SNGRGV (SEQ ID NO:488).29. The recombinant AAV vector of claim 14, wherein the capsid proteincomprises a substitution of amino acids 455-460 of SEQ ID NO:8 with thesequence SNGRGV (SEQ ID NO:488) and a substitution of amino acids494-501 of SEQ ID NO:8 with the sequence YPGGNYK (SEQ ID NO:491). 30.The recombinant AAV vector of claim 14, wherein the capsid proteincomprises a substitution of amino acids 455-460 of SEQ ID NO:8 with thesequence SNGRGV (SEQ ID NO:488) and a substitution of amino acids494-501 of SEQ ID NO:8 with the sequence KQKNVN (SEQ ID NO:492).
 31. Therecombinant AAV vector claim 14, wherein the heterologous polypeptide isa therapeutic polypeptide.
 32. The recombinant AAV vector of claim 14,wherein the heterologous RNA is a functional RNA.
 33. The recombinantAAV vector of claim 14, wherein the nucleic acid comprises terminalrepeat (TR) sequences from AAV2 or AAV4.